Targeting ligands for tau pathology

ABSTRACT

Methods and compositions for detecting tau pathology are described. The compositions for detecting tau pathology comprise a targeting ligand that specifically binds to a cell surface marker of tau pathology, wherein the targeting ligand is linked to a liposome that includes an imaging agent. The compositions can be used in a method for imaging tau pathology in a subject that comprises administering to the subject an effective amount of the composition to a subject and imaging at least a portion of the subject to determine if that portion of the subject exhibits tau pathology. The compositions can also be used to detect tau pathology in biological samples obtained from a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. patent applicationSer. No. 16/922,762, filed on Jul. 7, 2020, which claims the benefit ofU.S. Provisional Patent Application No. 62/871,380, filed on Jul. 8,2019. This application also claims the benefit of U.S. ProvisionalPatent Application No. 63/185,317, filed on May 6, 2021, and U.S.Provisional Patent Application No. 63/287,036, filed on Dec. 7, 2021.Each of these applications is incorporated by reference herein in itsentirety.

SEQUENCE LISTING

A Sequence Listing has been submitted electronically in ASCII format andis hereby incorporated by reference in its entirety. The ASCII copy,created on May 5, 2022, is named Alzeca-ADx-002-USCIP_ST25.txt and is5,659 bytes in size.

BACKGROUND

The microtubule associated protein tau, coded for by the MAPT gene, isabundant in the brain and is present in neurons, glia, and other celltypes. Tau expressed in six isoforms has a vast array ofpost-translational modifications, including glycosylation, glycation,nitration, ubiquitination, and more than 80 potential phosphorylationsites expanding the complexity of its role in health and disease. Adefinitive feature of many neurodegenerative diseases, includingAlzheimer's disease (AD), frontotemporal lobar degeneration (FTLD), andParkinson's disease (PD) (collectively termed “tauopathies”), is thepresence of intracellular aggregated filamentous tau.

The transition from physiological soluble tau to insoluble tau isprimarily associated with changes in its phosphorylation state leadingto oligomeric tau and tau fibrils known as paired helical fragments(PHF) that form characteristic neurofibrillary tangles (NFT). Tauaggregates are also capable of “infecting” a healthy cell, inducingfurther misfolding, aggregation, and neurotoxicity. Studies ofintercellular propagation demonstrate passage through an extracellularphase that progresses throughout the brain.

The National Institute of Aging-Alzheimer's Association (NIA-AA)Research Framework identifies extracellular deposits of amyloid beta(A), presence of intraneuronal hyperphosphorylated tau (T), and markersof neurodegeneration or neuronal injury (N) as characterization of AD.Each biomarker is scored either positive or negative. To be on the ADcontinuum, A+ (Amyloid positive) is required, while a positive diagnosisof AD requires A+ and T+. Biomarker detection can be by: (i) positronemission tomography (PET) imaging of amyloid and tau; (ii) cerebrospinalfluid (CSF) detection of reduced Aβ₄₂, and/or high Aβ₄₀/Aβ₄₂ and highphosphorylated tau and total tau; or (iii) neuronal injury ordegeneration as shown by structural brain magnetic resonance imaging(MRI). Tracking of brain pathology in longitudinal studies suggests thattau pathology may precede Aβ accumulation but is undetectable based oncurrent biomarker detection threshold levels and is amplifiedcatastrophically by independent AP deposition. The ATN researchframework-based diagnosis of AD is therefore limited by tau pathologydetection. Other factors to consider in the development of tau detectionmethods include the invasive nature of CSF sampling requiring lumbarpuncture, and in the case of PET imaging, exposure to ionizingradiation, high cost, well documented side effects, irregularavailability in primary care settings, and uneven geographicalavailability of PET scanners and isotopes. The short half-life of PETagents also poses challenges for the detection of intracellular tau inthe early stages of tau pathology formation. Blood based markers arevery promising, but only provide an indirect measure that cannot provideinformation on the localization of tau pathology in brain. Methods todetect early tau pathology that avoid these pitfalls are thereforehighly desirable.

SUMMARY

The initiation of tau pathology is marked by abnormal phosphorylation oftau. Hyperphosphorylative conditions in neurons may result in uniquesurface markers, which is consistent with an altered balance ofkinase-phosphatase activity resulting in elevated levels ofhyperphosphorylated tau species. The utility of such a surface markerlies in the fact that an imaging agent can bind it without needing topenetrate the cell membrane, a limitation that currently hinders tau-PETagents.

Reverse phase protein array (RPPA) analysis of a cell-based model of tauhyperphosphorylation identified several proteins that were eitherupregulated or downregulated by the onset of a hyperphosphorylativestate. Iterative Cell-SELEX process was used to identify DNAthioaptamers that specifically bind such cells. High T1 relaxivityPEGylated liposomes bearing macrocyclic Gd-chelates were modified tobear the thioaptamers on their surface, thus enabling targeting of theparticles to the surface of hyperphosphorylative cells forcontrast-enhanced MM.

Thus, in one aspect, a new generation of molecular imaging probes isprovided for in vivo detection of cells performing abnormalphosphorylation representing the initial stages of pTau formation,enabling a very early stage diagnosis of AD. In one aspect, a novelnanoparticle formulation that binds such abnormally phosphorylatingcells, enabling in vivo visualization of the hyperphosphorylative stateby MRI. The results demonstrate the potential of this novel platform todiagnose the development of future tau pathology and has implicationsfor the very early stage diagnosis of Alzheimer's disease.

At the molecular level, binding targets of the thioaptamers wereidentified, such as vimentin, a normally intracellular protein that isspecifically expressed on the surface of cells underhyperphosphorylative conditions, representing a possible biomarker ofthe pathological hyperphosphorylation found in AD. Cell-surface vimentinwas found to be present at elevated levels on SH-SY5Y and ReN-VM cellsin the hyperphosphorylative state showing elevated pTau levels. Suchcells were also found to be specifically bound by the thioaptamers. In 2month old P301S transgenic mice, elevated vimentin levels were found incells of the hippocampus that also had elevated pTau levels, butnon-transgenic siblings did not exhibit either elevated pTau orvimentin. To demonstrate vimentin as a specific target of the DNAthioaptamer, withaferin A, a small molecule vimentin ligand, was boundto Gd-containing liposomes. Both the thioaptamer targeted Gd-containingliposomes and the Withaferin targeted Gd-containing liposomes, wheninjected intravenously in P301S mice at 2 months of age, specificallyproduced signal enhancement on MRI in the brains of transgenic mice, butnot in the brains of non-transgenic siblings. Untargeted Gd-containingliposomes did not show any signal enhancement in either group of mice.Practically 100% of the transgenic mice go on to develop frank taupathology at 8 months of age or later. The targeted Gd-containingliposomes therefore serve as an M-MRI agent that can identify thedevelopment of future tau pathology in a pre-pathological state.

In one aspect, a composition is provided for identifying tau pathology,the composition comprising a targeting ligand that specifically binds toa cell surface marker of tau pathology, wherein the targeting ligand islinked to a liposome comprising an imaging agent, e.g., an MRI contrastenhancing agent. In some aspects, the targeting ligand comprises anaptamer or stabilized aptamer. In some aspects, the targeting ligandcomprises a thioaptamer. In some aspects, the targeting ligand comprisesa DNA nucleotide sequence selected from one or more of Tau_1 (SEQ ID NO:5) (sometimes referred to hereinafter as “DONGYBM”), Tau_3 (SEQ ID NO:6) (sometimes referred to hereinafter as “MUSQD”), Tau_9 (SEQ ID NO: 7),Tau_11 (SEQ ID NO: 8), Tau_10 (SEQ ID NO: 9), Tau_13 (SEQ ID NO: 10),Tau_8 (SEQ ID NO: 11), Tau_4 (SEQ ID NO: 12), Tau_17 (SEQ ID NO: 13),Tau_5 (SEQ ID NO: 14), Tau_21 (SEQ ID NO: 15), Tau_25 (SEQ ID NO: 16),Tau_7 (SEQ ID NO: 17), Tau_31 (SEQ ID NO: 18), Tau_42 (SEQ ID NO: 19),Tau_14 (SEQ ID NO: 20), Tau_19 (SEQ ID NO: 21), Tau_15 (SEQ ID NO: 22),Tau_56 (SEQ ID NO: 23), Tau_34 (SEQ ID NO: 24), Tau_23 (SEQ ID NO: 25),Tau_99 (SEQ ID NO: 26), and Tau_102 (SEQ ID NO: 27).

In some aspects, the cell surface marker of tau pathology comprises acell surface marker of tau hyperphosphorylation. In some aspects, thecell surface marker of tau pathology comprises a protein selected fromkeratin 6A (KRT6A), keratin 6B (KRT6B), heat shock protein (HSP), andvimentin (VIM). In some aspects, the targeting ligand is determined tospecifically bind to a cell surface marker of tau pathology using asystematic evolution of ligands by exponential enrichment (SELEX)method. In some aspects, the targeting ligand is linked to polyethyleneglycol that is conjugated to a phospholipid that associates with theliposome. In some aspects, the liposome comprises a membrane, themembrane comprising: a first phospholipid; a sterically bulky excipientthat is capable of stabilizing the liposome; a second phospholipid thatis derivatized with a first polymer; a third phospholipid that isderivatized with a second polymer, the second polymer being conjugatedto the targeting ligand; and an imaging agent that is encapsulated by orbound to the membrane.

In another aspect, a method is provided for imaging tau pathology in asubject, the method comprising: administering to the subject adetectably effective amount of a targeting ligand-liposome conjugatecomprising a targeting ligand that specifically binds to a cell surfacemarker of tau pathology, wherein the targeting ligand is conjugated to aliposome comprising an imaging agent, and imaging at least a portion ofthe subject to determine if that portion of the subject exhibits taupathology. In some aspects, the portion of the subject includes aportion of the subject's brain. In some aspects, the imaging indicates alevel of tau pathology sufficient to diagnose the subject as havingearly stage AD. In some aspects, the method further comprises providingprophylaxis or treatment of AD to the subject. In some aspects, theimaging agent is an MRI contrast enhancing agent, and the level ofbinding is determined using MRI.

In another aspect, a method is provided for detecting tau pathology, themethod comprising: contacting a biological sample with an effectiveamount of a targeting ligand-liposome conjugate comprising a targetingligand that specifically binds to a cell surface marker of taupathology, wherein the targeting ligand is conjugated to a liposomecomprising a detectable label; washing the biological sample to removeunbound targeting ligand liposome conjugate; and detecting tau pathologyin the biological sample by determining the amount of detectable labelremaining in the biological sample. In some aspects, the biologicalsample is a sample containing neural cells.

In another aspect, a targeting composition is provided, the targetingcomposition comprising: a phospholipid linked to a polymer that islinked to a targeting ligand that specifically binds to a cell surfacemarker of tau pathology. In some aspects, the targeting ligand is anaptamer or stabilized aptamer. In some aspects, the targeting ligand isa thioaptamer. In some aspects, the aptamer or stabilized aptamercomprises a DNA nucleotide sequence selected from one or more of Tau_1(SEQ ID NO: 5; DONGYBM), Tau_3 (SEQ ID NO: 6; MUSQD), Tau_9 (SEQ ID NO:7), Tau_11 (SEQ ID NO: 8), Tau_10 (SEQ ID NO: 9), Tau_13 (SEQ ID NO:10), Tau_8 (SEQ ID NO: 11), Tau_4 (SEQ ID NO: 12), Tau_17 (SEQ ID NO:13), Tau_5 (SEQ ID NO: 14), Tau_21 (SEQ ID NO: 15), Tau_25 (SEQ ID NO:16), Tau_7 (SEQ ID NO: 17), Tau_31 (SEQ ID NO: 18), Tau_42 (SEQ ID NO:19), Tau_14 (SEQ ID NO: 20), Tau_19 (SEQ ID NO: 21), Tau_15 (SEQ ID NO:22), Tau_56 (SEQ ID NO: 23), Tau_34 (SEQ ID NO: 24), Tau_23 (SEQ ID NO:25), Tau_99 (SEQ ID NO: 26), and Tau_102 (SEQ ID NO: 27).

In some aspects, an aptamer or stabilized aptamer is provided, theaptamer or stabilized aptamer comprising a DNA nucleotide sequenceselected from one or more of Tau_1 (SEQ ID NO: 5; DONGYBM), Tau_3 (SEQID NO: 6; MUSQD), Tau_9 (SEQ ID NO: 7), Tau_11 (SEQ ID NO: 8), Tau_10(SEQ ID NO: 9), Tau_13 (SEQ ID NO: 10), Tau_8 (SEQ ID NO: 11), Tau_4(SEQ ID NO: 12), Tau_17 (SEQ ID NO: 13), Tau_5 (SEQ ID NO: 14), Tau_21(SEQ ID NO: 15), Tau_25 (SEQ ID NO: 16), Tau_7 (SEQ ID NO: 17), Tau_31(SEQ ID NO: 18), Tau_42 (SEQ ID NO: 19), Tau_14 (SEQ ID NO: 20), Tau_19(SEQ ID NO: 21), Tau_15 (SEQ ID NO: 22), Tau_56 (SEQ ID NO: 23), Tau_34(SEQ ID NO: 24), Tau_23 (SEQ ID NO: 25), Tau_99 (SEQ ID NO: 26), andTau_102 (SEQ ID NO: 27).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows (A) SH SY5Y cells (B) when treated with 30 μM retinoicacid (RA) for 10 days to differentiate to a neuron-like phenotype withaxonal and dendritic structures. These cells develop long cell bodiesand begin to form neurite-like processes.

FIG. 1B shows the SH SY5Y cells after treatment with okadaic acid (OA),demonstrating that the cells avidly phosphorylate tau. The upper rowshows that the differentiated cells exhibit pTau Thr205/Ser202 (an earlyphase of phosphorylation) mostly in the perinuclear region. The lowerrow shows that after exposure to 30 nM OA for 24 h, the cells exhibitsignificant levels of pTau Ser396 (a late stage of phosphorylation)throughout the cytoplasm.

FIG. 1C shows the presence of p-tau396 in SHSY5Y cells inhyperphosphorylative conditions. The cells were differentiated with 30μM RA for 10 days and exposed to 30 nM OA for 24 h. Under thesehyperphosphorylative conditions, tau is over-expressed andhyperphosphorylated.

FIG. 2A shows a pictorial representation of the cell-SELEX process.

FIG. 2B shows the abundance of the top 23 sequences from SELEX cycle1-26 depicting their evolution. The fractions are low until about cycle10, when they increase sharply. The abundance of Tau_1 (SEQ ID NO: 5;DONGYBM) and Tau_3 (SEQ ID NO: 6; MUSQD) continually increase withincreasing cycle number.

FIG. 2C shows undifferentiated, differentiated (RA), andhyperphosphorylated (OA) SH-SY5Y cells stained with 50 nM Cy5 labelledTau_1 (SEQ ID NO: 5; DONGYBM) aptamer for 2 h at 4° C.

FIG. 2D shows Tau_1 (SEQ ID NO: 5; DONGYBM) and Tau_3 (SEQ ID NO: 6;MUSQD) secondary structures using Mfold.

FIG. 2E shows a cladogram showing the relationship between the top 23tau aptamer sequences and the aptamer families.

FIG. 3 shows the saturation binding curves generated using Cy5 labeledTau_1 (SEQ ID NO: 5; DONGYBM) and Tau_3 (SEQ ID NO: 6; MUSQD) afterserial dilution of aptamer solutions with target hyperphosphorylated andnon-target differentiated and undifferentiated SH-SY5Y and ReN-VM cells.

FIG. 4A shows an example liposomal-Gd nanoparticle contrast agent. Asshown, the liposomal bilayer incorporates DSPE-DOTA-Gd for MR contrast,lissamine rhodamine for fluorescence imaging, DSPEmPEG2000 to enhancecirculation half-life, DSPEPEG3400 for non-targeted (control/stealth)liposomes, and DSPE-PEG3400-aptamer (Tau_1 (SEQ ID NO: 5; DONGYBM) orTau_3 (SEQ ID NO: 6; MUSQD)) for targeted ADx-002 (or “TauX”)nanoparticles.

FIG. 4B shows the synthesis of lipidized Withaferin A (WNP).

FIG. 5A shows pre- and post-contrast images for T1-weighted spin echo(T1w-SE) and fast spin echo inversion recovery (FSE-IR), whichdemonstrate signal enhancement in delayed post-contrast scans oftransgenic (Tg) P301S mice treated with ADx-002 relative to age-matchedwild type (WT) controls. The Tg animals showed high enhancement incortical and hippocampal regions (as indicated by the arrows). The Tganimals show no signal enhancement four days after injection ofuntargeted contrast (UC).

FIG. 5B shows box and whisker plots demonstrating signal enhancement inTg animals relative to WT counterparts and UC-treated Tg animals forboth Tlw-SE and FSE-IR sequences (*p<0.05; **p<0.005). The dotted lineindicates the signal threshold for determining sensitivity (two standarddeviations above baseline noise, ˜6%).

FIG. 5C shows Receiver operating characteristic (ROC) curves plottingtrue positive fraction (TPF) against false positive fraction (FPF),demonstrating ADx-002 accuracy in identifying early age Tg animals. Afitted curve connects the observed operating points. Area under curve(AUC) is calculated using the fitted curve, and sensitivity (truepositive rate) and specificity (true negative rate) for bothformulations are listed.

FIG. 5D shows the results of immunofluorescence studies on brainsections harvested post MRI scans of P301S mice treated with ADx-002nanoparticles.

FIG. 6A shows the presence of the thioaptamers binding a target onSHSY5Y cells; undifferentiated and hyperphosphorylated OA (24 h, 30 nM)and QA (24 h, 100 nM) were co-stained with VIM (D21H3) and ptau (AT100)antibodies.

FIG. 6B shows the presence of the aptamers binding target on SHSY5Ycells; undifferentiated and hyperphosphorylated OA (24h, 30 nM) and QA(24h, 100 nM) co-stained with cell surface (CS) VIM (Clone 84-1)antibody and aptamer Tau_1 (SEQ ID NO: 5; DONGYBM (50 nM); nuclei werecounterstained with DAPI.

FIG. 6C shows expression of VIM in P301STG and WT frozen mouse tissuesections stained with VIM (SP20) and ptau (AT100) antibodies and DAPIstained nuclei.

FIG. 7 shows the results of an in vitro binding study of Withaferinnanoparticles. Differentiated and hyperphosphorylative SH SY5Y cellswere incubated with Rhodamine labeled Withaferin nanoparticles (WNP) for30 min at 37° C. Binding is specificity observed to cells underhyperphosphorylative conditions.

FIG. 8A shows pre- and post-contrast MRI images for T1-weighted spinecho (Tlw-SE) and fast spin echo inversion recovery (FSE-IR),demonstrating signal enhancement in delayed post-contrast scans of TgP301S mice and APP/PSEN1 mice treated with WNP relative to age-matchedWT controls.

FIG. 8B shows box and whisker plots demonstrating signal enhancement inTg P301S mice and APP/PSEN1 mice treated with WNP relative to WTcounterparts and UC-treated Tg animals for both Tlw-SE and FSE-IRsequences. The dotted line indicates the signal threshold fordetermining sensitivity (two standard deviations above baseline noise,˜6%).

FIG. 8C shows ROC curves plotting true positive TPF against FPF,demonstrating WNP accuracy in identifying early age Tg animals. A fittedcurve connects the observed operating points. AUC is calculated usingthe fitted curve, and sensitivity (true positive rate) and specificity(true negative rate) are listed.

FIG. 9 shows the phosphorylation status of tau in WNP treated P301Smice. P301S mice aged 2 months treated with WNP were sacrificedimmediately after MR image acquisition, and intact brains were recoveredfor immunofluorescence. Frozen brain tissue sections were stained withAT8 antibody. Positive signal on Tg mice sections but not on the WTconfirm the presence of phosphorylated tau in Tg mice.

FIG. 10 shows cell surface VIN expression on 2 month old P301S mice. Themice were perfused with heparin/PBS and formalin, and brains wereisolated and processed to immunolabel frozen sections with cell-surfaceVIM antibody (clone 84-1, Abnova) that specifically stains VIMtranslocated to the cell surface. Tg mice showed a higher expression incomparison with WT mice.

DETAILED DESCRIPTION

This disclosure provides methods and compositions for detecting taupathology. The compositions for detecting tau pathology comprise atargeting ligand that specifically binds to a cell surface marker of taupathology, wherein the targeting ligand is linked to a liposome thatincludes an imaging agent. The compositions may be used in a method forimaging tau pathology in a subject that comprises administering to thesubject an effective amount of the composition and imaging at least aportion of the subject to determine if that portion of the subjectexhibits tau pathology. The compositions may also be used to detect taupathology in biological samples obtained from a subject.

Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent specification, including definitions, will control.

Unless otherwise specified, “a,” “an,” “the,” “one or more of,” and “atleast one” are used interchangeably. The singular forms “a”, “an,” and“the” are inclusive of their plural forms.

The recitations of numerical ranges by endpoints include all numberssubsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3,3.80, 4, 5, etc.).

The term “about,” when referring to a value or to an amount of mass,weight, time, volume, concentration, or percentage is meant to encompassvariations of ±10% from the specified amount.

The terms “comprising” and “including” are intended to be equivalent andopen-ended.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.

The phrase “selected from the group consisting of” is meant to includemixtures of the listed group and combinations thereof.

An “effective” or a “detectably effective amount” of a composition meansan amount sufficient to detect the presence of cell surface markersassociated with tau pathology or to yield an acceptable image usingequipment that is available for clinical use. A detectably effectiveamount of a detecting or imaging agent may be administered in more thanone injection. The detectably effective amount of the detecting orimaging agent may vary according to factors such as the degree ofsusceptibility of the individual, the age, sex, and weight of theindividual, idiosyncratic responses of the individual, and thedosimetry. Detectably effective amounts of the detecting or imagingagent may also vary according to instrument and film-related factors.Optimization of such factors is well within the level of skill in theart. The amount of imaging agent used for diagnostic purposes, and theduration of the imaging study will depend upon the specific imagingagent used, the body mass of the patient, the nature and severity of thecondition being treated, the nature of therapeutic treatments underwhich the patient has gone, and on the idiosyncratic responses of thepatient. Ultimately, the attending physician will decide the amount ofimaging agent to administer to each individual patient and the durationof the imaging study.

The term “diagnosis” may encompass determining the nature of a diseasein a subject, as well as determining the severity and probable outcomeof the disease or episode of the disease, the prospect of recovery(prognosis), or both. “Diagnosis” may also encompass diagnosis in thecontext of rational therapy, in which the diagnosis guides therapy,including initial selection of therapy, modification of therapy (e.g.,adjustment of dose and/or dosage regimen), and the like.

The term antigen refers to a molecule or a portion of a molecule capableof being bound by a targeting ligand. An antigen is typically alsocapable of inducing an animal to produce an antibody capable of bindingto an epitope of that antigen. An antigen can have one or more than oneepitope. The specific reaction referred to above is meant to indicatethat the antigen will react, in a highly selective manner, with itscorresponding antibody and not with the multitude of other antibodiesthat can be evoked by other antigens.

The term epitope refers to that portion of any molecule capable of beingrecognized by, and bound by, a targeting ligand such as an aptamer. Ingeneral, epitopes comprise chemically active surface groupings ofmolecules, for example, amino acids or sugar side chains, and havespecific three-dimensional structural and specific chargecharacteristics.

The phrase “specifically binds” refers to a targeting ligand binding toa target structure, wherein the targeting ligand binds the targetstructure, or a sub-unit thereof, but does not bind to a biologicalmolecule that is not the target structure, or the targeting ligand atleast binds preferentially to the target structure. Targeting ligands(e.g., thioaptamers) that specifically bind to a target structure or asub-unit thereof may not cross-react with biological molecules that areoutside of the target structure family.

The term “polynucleotide” refers to a nucleic acid sequence includingDNA, RNA, and micro-RNA and can refer to markers that are eitherdouble-stranded or single-stranded. Polynucleotide can also refer tosynthetic variants with alternative sugars such as locked nucleic acids.

Compositions for Identifying Tau Pathology

In one aspect, a composition for identifying tau pathology is provided,the composition comprising a targeting ligand that specifically binds toa cell surface marker of tau pathology, wherein the targeting ligand islinked to a liposome comprising an imaging agent.

In some aspects, the cell surface marker of tau pathology is a cellsurface marker of tau hyperphosphorylation. Tau pathology refers toabnormal tau protein that results in taupathies. Tau pathology resultsfrom the hyperphosphorylation of tau protein. Normal tau contains 2-3mol phosphate/mol protein, whereas hyperphosphorylated tau proteinincludes substantially higher levels of phosphate. Hyperphosphorylatedtau leads to the formation of neurofibrillary tangles. Tau proteinexists within the cell and is difficult to detect directly. However,certain cell surface markers (i.e., epitopes) are associated with theunderlying tau pathology. In some aspects, these cell surface markersare epitopes that have been identified using the Cell-SELEX method, inwhich neurons exhibiting tau pathology or cell models of neurons areused as targets for target ligands (e.g., thioaptamers). In someaspects, the cell surface marker of tau pathology comprises a proteinselected from KRT6A, KRT6B, HSP, and VIM.

Targeting Ligands

The term “targeting ligand” as used herein includes any molecule thatcan be linked to the liposome for the purpose of engaging a specifictarget and, in particular, for recognizing tau pathology. Examples ofsuitable targeting ligands include, but are not limited to, antibodies,antibody fragments, thioaptamers, aptamers, and stabilized aptamers. Insome aspects, targeting ligands can be thioaptamers that specificallybind to cell surface markers for tau pathology.

The targeting ligands specifically bind to cells exhibiting taupathology. Specific binding refers to binding that discriminates betweenthe selected target and other potential targets and binds withsubstantial affinity to the selected target. Substantial affinityrepresents a targeting ligand having a binding dissociation constant ofat least about 10⁻⁸ mol/m³, but in other aspects, the targeting ligandcan have a binding dissociation constant of at least about 10⁻⁹ mol/m³,about 10⁻¹⁰ mol/m³, about 10⁻¹¹ mol/m³, or at least about 10⁻¹² mol/m³.

In some aspects, the targeting ligand is an aptamer. An aptamer is anucleic acid that binds with high specificity and affinity to aparticular target molecule or cell structure, through interactions otherthan Watson-Crick base pairing. Suitable aptamers may be single strandedRNA, DNA, a modified nucleic acid, or a mixture thereof. The aptamerscan also be in a linear or circular form. In some aspects, the aptamersare single stranded DNA, while in other aspects, they are singlestranded RNA.

Aptamer functioning is unrelated to the nucleotide sequence itself, butrather is based on the secondary/tertiary structure formed by thepolynucleotide, and aptamers are therefore best considered as non-codingsequences. Binding of a nucleic acid ligand to a target molecule is notdetermined by nucleic acid base pairing, but by the three-dimensionalstructure of the aptamer. In solution, the chain of nucleotides formsintramolecular interactions that fold the molecule into a complexthree-dimensional shape. The shape of the nucleic acid ligand allows itto bind tightly against the surface of its target molecule. In additionto exhibiting remarkable specificity, nucleic acid ligands generallybind their targets with very high affinity, e.g., the majority ofanti-protein nucleic acid ligands have equilibrium dissociationconstants in the femtomolar to low nanomolar range.

The length of the aptamers suitable for use as targeting ligands is notparticularly limited and includes aptamers including about 10 to about200 nucleotides, about 100 nucleotides or less, about 50 nucleotides orless, about 40 nucleotides or less, or about 35 nucleotides or less. Insome aspects, the aptamer has a size from about 15 to about 40nucleotides. In addition, in almost all known cases, the variousstructural motifs that are involved in the non-Watson-Crick type ofinteractions involved in aptamer binding, such as hairpin loops,symmetric and asymmetric bulges, and pseudoknots, can be formed innucleic acid sequences of 30 nucleotides or less.

In some aspects, the aptamers are stabilized aptamers that comprise achemical modification to increase their stability. Modificationsinclude, but are not limited to, those that provide other chemicalgroups that incorporate additional charge, polarizability,hydrophobicity, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,2-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil,backbone modifications, phosphorothioate or alkyl phosphatemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine, and the like. Modificationscan also include 3′ and 5′ modifications such as capping. In certainaspects, the nucleic acid ligands comprise RNA molecules that are2′-fluoro (2′-F) modified on the sugar moiety of pyrimidine residues.

Suitable stabilized aptamers can further include nucleotide analogs,such as, for example, xanthine or hypoxanthine, 5-bromouracil,2-aminopurine, deoxyinosine, or methylated cytosine such as5-methylcytosine, N4-methoxydeoxycytosine, and the like. Also includedare bases of polynucleotide mimetics, such as methylated nucleic acids,e.g., 2′-O-methRNA, peptide nucleic acids, locked nucleic acids,modified peptide nucleic acids, and any other structural moiety thatacts substantially like a nucleotide or base, for example, by exhibitingbase-complementarity with one or more bases that occur in DNA or RNA.

In some aspects, the stabilized aptamer comprises a thioaptamer.Thioaptamers are aptamers in which one or both of the non-bridgingoxygen atoms have been substituted with sulfur. Oxygen-to-sulfursubstitutions not only increases the stability of the aptamer, but insome cases also increases its binding affinity.

Typically, the targeting ligand (e.g., aptamer) is linked to a liposomecomprising an imaging agent. However, the aptamers themselves are noveland useful. Examples of suitable aptamers include those comprising a DNAnucleotide sequence selected from, and in some instances, selected fromthe group consisting of: Tau_1 (SEQ ID NO: 5), Tau_3 (SEQ ID NO: 6),Tau_9 (SEQ ID NO: 7), Tau_11 (SEQ ID NO: 8), Tau_10 (SEQ ID NO: 9),Tau_13 (SEQ ID NO: 10), Tau_8 (SEQ ID NO: 11), Tau_4 (SEQ ID NO: 12),Tau_17 (SEQ ID NO: 13), Tau_5 (SEQ ID NO: 14), Tau_21 (SEQ ID NO: 15),Tau_25 (SEQ ID NO: 16), Tau_7 (SEQ ID NO: 17), Tau_31 (SEQ ID NO: 18),Tau_42 (SEQ ID NO: 19), Tau_14 (SEQ ID NO: 20), Tau_19 (SEQ ID NO: 21),Tau_15 (SEQ ID NO: 22), Tau_56 (SEQ ID NO: 23), Tau_34 (SEQ ID NO: 24),Tau_23 (SEQ ID NO: 25), Tau_99 (SEQ ID NO: 26), and Tau_102 (SEQ ID NO:27).

In some aspects, the aptamers are positioned between two primernucleotide sequences that facilitate amplification of the aptamersequence, e.g., by Polymerase Chain Reaction (PCR). For example, in someaspects, the DNA nucleotide sequence of the aptamer is positionedbetween the sequences GATATGTCTAGAGCCTCAGATCA (SEQ ID NO: 1) andCGGAGTTATGTTAGCAGTAGC (SEQ ID NO: 2). In other aspects, the DNAnucleotide sequence of the aptamer is positioned between the sequencesCGC TCG ATA GAT CGA GCT TCG (SEQ ID NO: 3) and GTC GAT CAC GCT CTA GAGCAC (SEQ ID NO: 4).

Validation of Cell Surface Changes in Hyperphosphorylative Conditions

SH-SY5Y, a human neuroblastoma cell line that can be differentiated intoneuron-like cells by changes in culture medium, was used to modelcell-surface changes under hyperphosphorylative conditions. As depictedin FIGS. 1A-1C, RA was used to induce cell differentiation marked bytemporal changes in morphology including the formation and lengtheningof neurites and with a strong increase in levels of intracellular tau.Imbalance in the kinase and phosphatase activity leading tohyperphosphorylation, simulating early stages of tauopathies, wasinduced by the use of a cell permeable neurotoxin OA (30 nM, 24 h) andconfirmed by the increase in phosphorylated tau S396 (see FIGS. 1B and1C). In parallel experiments, a milder agent, excitotoxin quinolinicacid (QA) 1 μM, was used to induce hyperphosphosphorylation.

RPPA analysis conducted on lysates of both RA-treated and untreatedSH-SY5Y cells rendered hyperphosphorylative by either OA (30 nM, 24 h)or QA (1 μM, 24 h) demonstrated marked changes in hyperphosphorylativecells. Ninety-eight unphosphorylated proteins and 36 phosphorylatedproteins were tested that showed significant change in their expressionunder different conditions. Uniprot protein associations showed that 44cell-membrane associated proteins, 10 peripheral membrane, and 12single-pass membrane proteins were significantly altered underhyperphosphorylative conditions. In summary, RPPA analysis demonstratedmarked cell surface changes in hyperphosphorylative cells includingover-expression of cell surface receptors.

Screening for Aptamers that Bind Cells in a Hyperphosphorylative State

Aptamer screening was performed using the cell-SELEX approach ondifferentiated SH-SY5Y cells in a hyperphosphorylative state. FIGS.2A-2E describe how cell-SELEX was used to identify biomarkers of onsetthe of AD. OA treated differentiated SH-SY5Y cells were used as asurrogate for hyperphosphorylative neurons to screen DNA aptamers thatspecifically recognize the differences between the surfaces of treatedand untreated cells, using the cell-SELEX methodology modified tocapture membrane binding aptamers. A total of 26 cell SELEX cycles wereperformed. To remove thioaptamers that bound common cell surfacemolecules not specific to the hyperphosphorylative state, a negativeselection was introduced at cycles 12 and 13 using differentiated,non-hyperphosphorylative cells (i.e., without OA treatment).Anticipating that selected thioaptamers would be systemically deliveredas nanoparticle imaging agents, the primary toxicity of which is drivenby hepatocyte uptake, another round of negative selection was conductedat cycles 20 and 21 using a hepatocyte cell-line THLE-3 to removeoligonucleotides that exhibited enhanced uptake by hepatocytes.

Tau_1 and Tau_3 Aptamers Specifically Bind Hyperphosphorylative Cells

Sequencing of all the selected pools using the Ion Torrent sequencingplatform revealed the evolution of families of DNA sequences, withenrichment particularly evident after 10 rounds of SELEX. Negativeselection eliminated certain sequences that were not specific to thehyperphosphorylative state or have a propensity for hepatocyte uptake.However, the relative abundance of key sequences increased steadilythroughout the whole process. The 23 most abundant sequences at round 26were identified, and their abundance throughout the SELEX process ascalculated using AptaAligner is shown in FIG. 2B. The sequence Tau_1(SEQ ID NO: 5; DONGYBM) was the most prevalent at cycle 26, representing20.6% of the thioaptamers present. A single base difference from thissequence, Tau_3 (SEQ ID NO: 6; MUSQD), was the second most representedsequence (10.4%). Binding studies using Cy5-labeled Tau_1 (SEQ ID NO: 5;DONGYBM) incubated with 99 undifferentiated, differentiated, anddifferentiated-hyperphosphorylative SH-SY5Y cells demonstrated elevatedbinding levels with the hyperphosphorylative cells (FIG. 2C). Thesecondary structure of the aptamers Tau_1 (SEQ ID NO: 5; DONGYBM) andTau_3 (SEQ ID NO: 6; MUSQD) calculated using mfold is shown in FIG. 2D.The sequences present at the final round were grouped by hierarchicalclustering and sequence homology using the multiple sequence alignmentcode MAFFT showing five distinct families, which were also presented asa cladogram (using the Clustal Omega) showing the common ancestrybetween these five aptamers families (FIG. 2E).

The apparent equilibrium dissociation constants (Kd_(app)) were measuredby serial dilution of aptamer solutions with target hyperphosphorylated,non-target differentiated, and undifferentiated SH-SY5Y cells. Theaffinity of these aptamers was also tested with another immortal neuralprogenitor stem cell line, ReN-VM, in hyperphosphorylative andnon-hyperphosphorylative conditions. The Kd_(app) for Tau_1 (SEQ ID NO:5; DONGYBM) and Tau_3 (SEQ ID NO: 6; MUSQD) with hyperphosphorylated SHSY5Y cells is 0.167±0.015 nM and 0.194±0.032 nM; and for the ReN-VMcells 318.15±46.2 nM and 234.24±38.6 nM, respectively (FIG. 3).

Sequencing

After identification, the aptamers may be sequenced. Sequencing may beby any method known in the art. DNA sequencing techniques includeclassic dideoxy sequencing reactions (Sanger method) using labeledterminators or primers and gel separation in slab or capillary,sequencing by synthesis using reversibly terminated labeled nucleotides,pyrosequencing, the 454 sequencing method, allele specific hybridizationto a library of labeled oligonucleotide probes, sequencing by synthesisusing allele specific hybridization to a library of labeled clones thatis followed by ligation, real time monitoring of the incorporation oflabeled nucleotides during a polymerization step, polony sequencing, andSOLiD sequencing. Sequencing may be by any method known in the art. Seefor example Sanger et al. (Proc Natl Acad Sci USA, 74(12): 5463 67,1977), Maxam et al. (Proc. Natl. Acad. Sci., 74: 560-564, 1977), andDrmanac, et al. (Nature Biotech., 16:54-58, 1998), which referencesdescribe example conventional ensemble sequencing techniques. Also seeLapidus et al. (U.S. Pat. No. 7,169,560), Quake et al. (U.S. Pat. No.6,818,395), Harris (U.S. Pat. No. 7,282,337), Quake et al. (U.S. patentapplication number 2002/0164629), and Braslaysky, et al., (PNAS (USA),100: 3960-3964, 2003), which references describe example single moleculesequencing by synthesis techniques. The contents of each of thesereferences is incorporated by reference herein in its entirety.

Several aptamers are disclosed herein that specifically bind to taupathology. Examples of these aptamers are described in Table 1.Accordingly, in some aspects, the aptamer or stabilized aptamercomprises a DNA nucleotide sequence selected from, including selectedfrom the group consisting of: Tau_1 (SEQ ID NO: 5; DONGYBM), Tau_3 (SEQID NO: 6; MUSQD), Tau_9 (SEQ ID NO: 7), Tau_11 (SEQ ID NO: 8), Tau_10(SEQ ID NO: 9), Tau_13 (SEQ ID NO: 10), Tau_8 (SEQ ID NO: 11), Tau_4(SEQ ID NO: 12), Tau_17 (SEQ ID NO: 13), Tau_5 (SEQ ID NO: 14), Tau_21(SEQ ID NO: 15), Tau_25 (SEQ ID NO: 16), Tau_7 (SEQ ID NO: 17), Tau_31(SEQ ID NO: 18), Tau_42 (SEQ ID NO: 19), Tau_14 (SEQ ID NO: 20), Tau_19(SEQ ID NO: 21), Tau_15 (SEQ ID NO: 22), Tau_56 (SEQ ID NO: 23), Tau_34(SEQ ID NO: 24), Tau_23 (SEQ ID NO: 25), Tau_99 (SEQ ID NO: 26), andTau_102 (SEQ ID NO: 27). In a further aspect, the aptamer or stabilizedaptamer comprises the DNA nucleotide sequence Tau_1 (SEQ ID NO: 5;DONGYBM), Tau_3 (SEQ ID NO: 6; MUSQD), or both.

TABLE 1 Aptamers that Specifically Bind to Tau Pathology IdentifierNucleotide Sequence SEQ ID NO Tau_1 CCCCCCACGGTCTCCGCTCCACA SEQ ID NO: 5AGTTCAC Tau_3 CCCCCCACGGTCTCCGCTCCACA SEQ ID NO: 6 AGTCCAC Tau_9CCCCCCACGGTCTCCGCTCCACA SEQ ID NO: 7 GGTTCAC Tau_11CCCCCCCACGGTCTCCGCTCCAC SEQ ID NO: 8 AAGTTCA Tau_10CTCGTGGGTGTGTGGTGGTGTTG SEQ ID NO: 9 TTGTGTG Tau_13CCCCCCACGGTCTCCGCTCCACA SEQ ID NO: 10 AGCCCAC Tau_8CTCGTCCCACCACAACATCATCT SEQ ID NO: 11 CAACGCC Tau_4CTCGTCCCACCACAACATTATCT SEQ ID NO: 12 CAACGCC Tau_17CTCGTGGGTGTACGGTGGTGTTG SEQ ID NO: 13 TTGTGTG Tau_5CTCCGACGGGATGTTCGATGAGC SEQ ID NO: 14 ACACACT Tau_21CCCCCCCACGGTCTCCGCTCCAC SEQ ID NO: 15 AAGTCCA Tau_25CCCCCCACGGTCTCCGCTCCACA SEQ ID NO: 16 GGTCCAC Tau_7CCCCCATTGGCTCCGCTCCACAC SEQ ID NO: 17 AGCTTCA Tau_31CCCCCCACGGTCTCCGCTCCACA SEQ ID NO: 18 AGCTCAC Tau_42CCCCCCCACGGTCTCCGCTCCAC SEQ ID NO: 19 AGGTTCA Tau_14CTCGTCCCACCACAACATTGTCT SEQ ID NO: 20 CAACGCC Tau_19CTCGTCCCACCACAACACCATCT SEQ ID NO: 21 CAACGCC Tau_15CTCCGACGGGGTGTTCGATGAGC SEQ ID NO: 22 ACACACT Tau_56CCCCCCGCGGTCTCCGCTCCACA SEQ ID NO: 23 AGTTCAC Tau_34TGGGTGTGTGGTGGTGTTGTTGT SEQ ID NO: 24 GTGGGTG Tau_23CTCGCCCCACCACAACATCATCT SEQ ID NO: 25 CAACGCC Tau_99CCCCCCACGGTCTCCGCTCCACA SEQ ID NO: 26 AGTTCGC Tau_102CCCCCCCACGGTCTCCGCTCCAC SEQ ID NO: 27 AAGCTCA

Targeting Ligand Conjugates

In some aspects, the targeting ligands (e.g., aptamers) are linked to aliposome or other vehicles for targeted delivery of an imaging ordetecting agent. For example, imaging or detecting agents can beencapsulated within the liposome. Employing such techniques, the taupathology-specific aptamers conjugated to a liposomal vesicle canprovide targeted delivery of imaging or detecting agents to cellsexpressing tau pathology. In some aspects, a single targeting ligand islinked to a liposome. In other aspects, targeting ligands (e.g., Tau_1(SEQ ID NO: 5; DONGYBM) and Tau_3 (SEQ ID NO: 6; MUSQD)) are linked tothe liposome.

The term “liposome” as used herein indicates a vesicular structurecomprised of lipids. The lipids typically have a tail group comprising along hydrocarbon chain and a hydrophilic head group. The lipids arearranged to form a lipid bilayer (i.e., membrane) with an inner aqueousenvironment suitable to contain an agent (e.g., imaging agent) to bedelivered. Such liposomes present an outer surface that may comprisesuitable targeting ligands that specifically bind to cell surfacemarkers of tau pathology. A suitable liposome platform may be, forexample, the “ADx” platform from Alzeca Biosciences, comprisinghydrogenated soy L-α-phosphatidylcholine (HSPC), cholesterol (Chol),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy (polyethyleneglycol)-2000) (DSPE-mPEG2000), and Gd(III)-DSPE-DOTA (the macrocyclicgadolinium imaging moiety, Gd(III)-DOTA, conjugated to a phospholipid,1,2-Distearoyl-sn-glycero-3-phosphorylethanolamine, DSPE), as well asthe entity used to conjugate the targeting ligand, DSPE-PEG-3400(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-3400]). See, e.g., U.S. Pat. No. 11,116,854, InternationalPatent Publication No. WO2020154623A1, and International PatentPublication No. WO2021163585A1, each of which is incorporated byreference herein in its entirety.

In some aspects, the membrane of the liposome may comprise at leastthree types of phospholipids. The membrane may comprise a firstphospholipid, which may be unmodified. Suitable first phospholipidsinclude those disclosed in U.S. Pat. Nos. 7,785,568 and 10,537,649, eachof which is incorporated by reference herein in its entirety. In oneaspect, the first phospholipid is HSPC. The membrane may include asecond phospholipid that may be derivatized with a first polymer.Suitable polymer-derivatized second phospholipids include thosedisclosed in U.S. Pat. Nos. 7,785,568 and 10,537,649. In one aspect, thesecond phospholipid that is derivatized with a first polymer isDSPE-mPEG2000. The membrane may include a third phospholipid that isderivatized with a second polymer, the second polymer ultimately beingconjugated to the targeting ligand. Suitable polymer-derivatized thirdphospholipids include those disclosed in U.S. Pat. Nos. 7,785,568 and10,537,649. One aspect, the third phospholipid that is derivatized witha second polymer is DSPE-PEG-3400.

In some aspects, the membrane may comprise a sterically bulky excipientthat is capable of stabilizing the liposome. Suitable excipients includethose disclosed in U.S. Pat. Nos. 7,785,568 and 10,537,649. In oneaspect, the sterically bulky excipient that is capable of stabilizingthe liposome is cholesterol.

In some aspects, the phospholipid moiety in thephospholipid-polymer-targeting ligand conjugate may be represented bythe following structural formula:

The variable m may be one of: 12, 13, 14, 15, 16, 17, or 18. Forexample, m may be 14 or 16. In various aspects, the phospholipid moietyin any of the first phospholipid, the second phospholipid, and thephospholipid-polymer-targeting ligand conjugate may be one of: HSPC,DPPC, DSPE, DSPC, or DPPE.

In some aspects, the polymer moiety in thephospholipid-polymer-targeting ligand conjugate is a polyol. Structuralunits forming polymers containing polyols comprise monomeric polyolssuch as pentaerythritol, ethylene glycol, and glycerin. Example polymerscontaining polyols comprise polyesters, polyethers, and polysaccharides.Example suitable polyethers include, but are not limited to, diols, suchas diols with the general formula HO—(CH2CH2O)p-H with p≥1, for example,polyethylene glycol, polypropylene glycol, and poly(tetramethyleneether) glycol. Suitable polysaccharides include, but are not limited to,cyclodextrins, starch, glycogen, cellulose, chitin, and beta-Glucans.Suitable polyesters include, but are not limited to, polycarbonate,polybutyrate, and polyethylene terephthalate, all terminated withhydroxyl end groups. Example polymers containing polyols comprisepolymers of about 500,000 Da or less molecular weight, including fromabout 300 to about 100,000 Da.

In some aspects, the polymer moiety in thephospholipid-polymer-targeting ligand conjugate comprises a hydrophilicpoly(alkylene oxide) polymer. The hydrophilic poly(alkylene oxide) mayinclude between about 10 and about 100 repeat units, and having, e.g., amolecular weight ranging from about 500-10,000 Da. The hydrophilicpoly(alkylene oxide) may comprise, for example, poly(ethylene oxide),poly (propylene oxide), and the like. The polymer moiety in thephospholipid-polymer-targeting ligand conjugate may be conjugated to thephospholipid moiety via an amide or carbamate group. The polymer moietyin the phospholipid-polymer-targeting ligand conjugate may be conjugatedvia an amide, carbamate, poly (alkylene oxide), triazole, combinationsthereof, and the like. For example, the polymer moiety in thephospholipid-polymer-targeting ligand conjugate may be represented byone of the following structural formulas:

The variable n may be any integer from about 10 to about 100, forexample, about 60 to about 100, about 70 to about 90, about 75 to about85, or about 77.

In some aspects, the phospholipid-polymer moiety in thephospholipid-polymer-targeting ligand conjugate may be represented byone of the following structural formulas:

The variable n may be any integer from about 10 to about 100, forexample, about 60 to about 100, about 70 to about 90, about 75 to about85, or about 77. The variable m may be one of: 12, 13, 14, 15, 16, 17,or 18. For example, n may be 77 and m may be 14. In another example, nmay be 77 and m may be 16.

In some aspects, the third phospholipid that is derivatized with asecond polymer, the second polymer being conjugated to the targetingligand, may comprise:

or a salt (e.g., an ammonium phosphate salt) thereof. In some aspects,the variable n may be any integer from about 10 to about 100, forexample, about 60 to about 100, about 70 to about 90, about 75 to about85, about 77, or about 79. The variable m may be one of: 12, 13, 14, 15,16, 17, or 18. For example, n may be 77, and m may be 14; n may be 79,and m may be 14; n may be 77, and m may be 16; and n may be 79, and mmay be 16.

The targeting ligands (e.g., aptamers) may be connected to one or morepolymer (e.g. PEG) moieties of the phospholipid-polymer-targeting ligandconjugate, with or without one or more linkers. The PEG moieties may beany type of PEG moiety (linear, branched, multiple branched, starshaped, comb shaped, or a dendrimer) and have any molecular weight. Thesame or different linkers or no linkers may be used to connect the sameor different PEG moieties to an aptamer. Commonly known linkers include,but are not limited to, amines, thiols, and azides, and can include aphosphate group. For example, in some aspects, the targeting ligand islinked to polyethylene glycol that is conjugated to a phospholipid thatassociates with the liposome.

In some aspects, the liposomes include a membrane, the membranecomprising: a first phospholipid selected from HSPC, DPPC, DSPE, DSPC,and DPPE; cholesterol; DPPC, DSPE, DSPC, and/or DPPE derivatized withPEG; DPPC, DSPE, DSPC, and/or DPPE derivatized with PEG and a targetingligand that specifically binds to a cell surface marker of taupathology; and an imaging agent that is encapsulated by or bound to themembrane. In further aspects, the targeting ligand is a thioaptamer, andthe imaging agent is an MM contrast enhancing agent.

In some aspects, a targeting composition is provided. In some aspects,the targeting composition includes a phospholipid linked to a polymerthat is linked to a targeting ligand that specifically binds to a cellsurface marker of tau pathology. The phospholipid can be any of thephospholipids described herein. In some aspects, the phospholipidcomprises one or more of DPPC, DSPE, DSPC, and DPPE. Likewise, thepolymer can be any of the polymers (e.g., polyols) described herein. Insome aspects, the polymer is polyethylene glycol.

Imaging or Detecting Agents

The composition for detecting tau pathology described herein may includean imaging or detecting agent. The imaging or detecting agent isgenerally associated with the liposome portion of the composition. Theimaging or detecting agent can be held within the liposome, or it can beconjugated to the liposome. In one aspect, the imaging or detectingagent is linked to a polymer that is linked to a phospholipid thatassociates with the membrane forming the liposome.

The liposomal composition comprises a macrocyclic Gd-based imagingagent. In some aspects, the macrocyclic gadolinium-based imaging agentcomprises Gd(III)-DOTA conjugated to a phospholipid, e.g.:

or a salt (e.g., a sodium salt) thereof. In some aspects, the variable xmay be one of: 12, 13, 14, 15, 16, 17, or 18. In one aspect, thevariable x is 16 and the conjugate is Gd(III)-DOTA-DSPE. Preparation ofGd(III)-DOTA-DSPE is described in U.S. Pat. No. 11,116,854.

In other aspects, the macrocyclic gadolinium-based imaging agentcomprises:

In one aspect, the imaging or detecting agent is linked to a polymerthat is linked to a phospholipid that associates with the membraneforming the liposome comprises Gd(III)-DOTA-DSPE.

In some aspects, the composition for detecting tau pathology includes adetecting agent. Examples of detecting agents include GFP, biotin,cholesterol, dyes such as fluorescence dyes, electrochemically activereporter molecules, and compositions comprising radioactive residues,such as radionuclides suitable for PET (positron emission tomography)detection, e.g., 18F, 11C, 13N, 15O, 82Rb or 68Ga.

In some aspects, the composition for detecting tau pathology comprisesan imaging agent. Imaging agents differ from detecting agents in thatthey not only indicate the presence of tau pathology but are suitablefor use with imaging methods that allow an image of a region of tissueexhibiting the tau pathology to be created and displayed. Examples ofimaging agents include near infrared imaging agents, positron emissiontomography imaging agents, single-photon emission tomography agents,fluorescent compositions, radioactive isotopes, and MM contrast agents.

In some aspects, the imaging agent is an MRI contrast enhancing agent.Disease detection using MM is often difficult because areas of diseasehave similar signal intensity compared to surrounding healthy tissue. Inthe case of MM, the imaging agent can also be referred to as a contrastagent. The MRI contrast enhancing agent may be a nonradioactive MRIcontrast enhancing agent that may be at least one of encapsulated by orbound to the membrane. For example, the nonradioactive MRI contrastenhancing agent may be both encapsulated by and bound to the membrane,e.g., to provide a dual contrast agent liposome. The liposomalcomposition may be characterized by a per-particle relaxivity in mM-1s-1 of at least about one or more of about: 100,000, 125,000, 150,000,165,000, 180,000, 190,000, and 200,000. Detecting the liposomalformulation may include detecting using MRI in a magnetic field rangeof, for example, between about 1T to about 3.5T, or about 1.5 to about3T. The nonradioactive MM contrast enhancing agent may includegadolinium. Suitable nonradioactive MM contrast enhancing agent mayinclude Gd(III)-DOTA-DSPE and (diethylenetriaminepentaaceticacid)-bis(stearylamide), gadolinium salt (Gd-DTPA-BSA). Gadoliniumparamagnetic chelates such as GdDTPA, GdDOTA, GdHPDO3A, GdDTPA-BMA, andGdDTPA-BSA are also suitable known MM contrast agents. See U.S. Pat. No.5,676,928 issued to Klaveness et al., which is incorporated by referenceherein in its entirety.

Methods of Imaging or Detecting Tau Pathology

In another aspect, a method is provided for imaging tau pathology in asubject. The method comprises administering to the subject a detectablyeffective amount of a targeting ligand-liposome conjugate comprising atargeting ligand that specifically binds to a cell surface marker of taupathology, wherein the targeting ligand is conjugated to a liposomecomprising an imaging agent, and imaging at least a portion of thesubject to determine if that portion of the subject exhibits taupathology.

In some aspects, a method for imaging tau pathology in a subject isprovided, the method comprising: (i) administering to the subject adetectably effective amount of a targeting ligand-liposome conjugatecomprising a targeting ligand that specifically binds to a cell surfacemarker of tau pathology, wherein the targeting ligand is conjugated to aliposome comprising an imaging agent; and (ii) imaging at least aportion of the subject to determine if the portion exhibits taupathology. The targeting ligand may comprise an aptamer. The targetingligand may comprise a stabilized aptamer. The targeting ligand maycomprise a thioaptamer. The targeting ligand may comprise a DNAnucleotide sequence selected from the group consisting of Tau_1 (SEQ IDNO: 5; DONGYBM), Tau_3 (SEQ ID NO: 6; MUSQD), Tau_9 (SEQ ID NO: 7),Tau_11 (SEQ ID NO: 8), Tau_10 (SEQ ID NO: 9), Tau_13 (SEQ ID NO: 10),Tau_8 (SEQ ID NO: 11), Tau_4 (SEQ ID NO: 12), Tau_17 (SEQ ID NO: 13),Tau_5 (SEQ ID NO: 14), Tau_21 (SEQ ID NO: 15), Tau_25 (SEQ ID NO: 16),Tau_7 (SEQ ID NO: 17), Tau_31 (SEQ ID NO: 18), Tau_42 (SEQ ID NO: 19),Tau_14 (SEQ ID NO: 20), Tau_19 (SEQ ID NO: 21), Tau_15 (SEQ ID NO: 22),Tau_56 (SEQ ID NO: 23), Tau_34 (SEQ ID NO: 24), Tau_23 (SEQ ID NO: 25),Tau_99 (SEQ ID NO: 26), and Tau_102 (SEQ ID NO: 27). The portion mayinclude a portion of the subject's brain. The imaging may indicate alevel of tau pathology sufficient to diagnose the subject as havingearly stage Alzheimer's disease. The imaging agent may be an MM contrastenhancing agent, and the level of binding may be determined using Mill.The cell surface marker of tau pathology may comprise a protein selectedfrom KRT6A, KRT6B, HSP, and VIM. The liposome may comprise a membrane,the membrane comprising: a first phospholipid; a sterically bulkyexcipient that is capable of stabilizing the liposome; a secondphospholipid that is derivatized with a first polymer; a thirdphospholipid that is derivatized with a second polymer, the secondpolymer being conjugated to the targeting ligand; and the imaging agent,which is encapsulated by or bound to the membrane.

In some aspects, a method for detecting tau pathology is provided, themethod comprising: contacting a biological sample with an effectiveamount of a targeting ligand-liposome conjugate comprising a targetingligand that specifically binds to a cell surface marker of taupathology, wherein the targeting ligand is conjugated to a liposomecomprising a detectable label; washing the biological sample to removeunbound targeting ligand liposome conjugate; and detecting tau pathologyin the biological sample by determining the amount of detectable labelremaining in the biological sample. The biological sample may compriseneural cells. The targeting ligand may comprise an aptamer. Thetargeting ligand may comprise a stabilized aptamer. The targeting ligandmay comprise a thioaptamer. The targeting ligand may comprise a DNAnucleotide sequence selected from the group consisting of Tau_1 (SEQ IDNO: 5; DONGYBM), Tau_3 (SEQ ID NO: 6; MUSQD), Tau_9 (SEQ ID NO: 7),Tau_11 (SEQ ID NO: 8), Tau_10 (SEQ ID NO: 9), Tau_13 (SEQ ID NO: 10),Tau_8 (SEQ ID NO: 11), Tau_4 (SEQ ID NO: 12), Tau_17 (SEQ ID NO: 13),Tau_5 (SEQ ID NO: 14), Tau_21 (SEQ ID NO: 15), Tau_25 (SEQ ID NO: 16),Tau_7 (SEQ ID NO: 17), Tau_31 (SEQ ID NO: 18), Tau_42 (SEQ ID NO: 19),Tau_14 (SEQ ID NO: 20), Tau_19 (SEQ ID NO: 21), Tau_15 (SEQ ID NO: 22),Tau_56 (SEQ ID NO: 23), Tau_34 (SEQ ID NO: 24), Tau_23 (SEQ ID NO: 25),Tau_99 (SEQ ID NO: 26), and Tau_102 (SEQ ID NO: 27). The method mayfurther comprise the step of obtaining the biological sample from asubject.

The term “subject” refers to an animal such as a vertebrate orinvertebrate animal. In some aspects, the subject is a mammal,including, but not limited to, primates, including simians and humans,equines (e.g., horses), canines (e.g., dogs), felines, variousdomesticated livestock (e.g., ungulates, such as swine, pigs, goats,sheep, and the like), as well as domesticated pets and animalsmaintained in zoos. In some aspects, the subject is a human subject. Insome aspects, the subject is a subject having an increased risk ofdeveloping AD. Risk factors for Alzheimer's disease include geneticpredisposition, smoking, diabetes, a history of head injuries,depression, and hypertension. See Burns A, Iliffe S., BMJ., 338: b158(2009)

The targeting ligand-liposome conjugate can include any of the featuresdescribed herein. For example, in some aspects, the targeting ligand isan aptamer or stabilized aptamer, while in further aspects, thetargeting ligand is a thioaptamer. In yet further aspects, the aptameror stabilized aptamer used in the method comprises a DNA nucleotidesequence selected from, including selected from the group consisting ofTau_1 (SEQ ID NO: 5; DONGYBM), Tau_3 (SEQ ID NO: 6; MUSQD), Tau_9 (SEQID NO: 7), Tau_11 (SEQ ID NO: 8), Tau_10 (SEQ ID NO: 9), Tau_13 (SEQ IDNO: 10), Tau_8 (SEQ ID NO: 11), Tau_4 (SEQ ID NO: 12), Tau_17 (SEQ IDNO: 13), Tau_5 (SEQ ID NO: 14), Tau_21 (SEQ ID NO: 15), Tau_25 (SEQ IDNO: 16), Tau_7 (SEQ ID NO: 17), Tau_31 (SEQ ID NO: 18), Tau_42 (SEQ IDNO: 19), Tau_14 (SEQ ID NO: 20), Tau_19 (SEQ ID NO: 21), Tau_15 (SEQ IDNO: 22), Tau_56 (SEQ ID NO: 23), Tau_34 (SEQ ID NO: 24), Tau_23 (SEQ IDNO: 25), Tau_99 (SEQ ID NO: 26), and Tau_102 (SEQ ID NO: 27).

In some aspects, a method is provided for generating an image of atissue region of a subject, by administering to the subject a detectablyeffective amount of the composition for detecting tau pathology, andgenerating an image of a portion of the subject (i.e., a tissue region)to which the composition including the imaging agent has distributed. Togenerate an image of the tissue region, it is necessary for a detectablyeffective amount of imaging agent to reach the tissue region ofinterest, but it is not necessary that the imaging agent be localized inthis region alone. However, in some aspects, the compositions includingthe imaging agents are targeted or administered locally such that theyare present primarily in the tissue region of interest. Examples ofimages include two-dimensional cross-sectional views andthree-dimensional images. In some aspects, a computer is used to analyzethe data generated by the imaging agents to generate a visual image. Thetissue region or portion of the subject can be an organ of a subjectsuch as the brain, heart, lungs, or blood vessels. In other aspects, theportion of the subject can be a tissue region known to include neuralcells, such as the brain. Examples of imaging methods include opticalimaging, fluorescence imaging, computed tomography, positron emissiontomography, single photon emission computed tomography, and MM. Anyother suitable type of imaging methodology known by those skilled in theart is contemplated.

In some aspects, the imaging agent is an MRI contrast enhancing agent,and the level of binding is determined using MRI. MRI is a medicalapplication of nuclear magnetic resonance and forms pictures of theanatomy and physiological processes of the body using strong magneticfields, magnetic field gradients, and radio waves to generate images ofa portion of a subject. MRI is commonly used for neuroimaging,cardiovascular imaging, musculoskeletal imaging, liver imaging, andgastrointestinal imaging. MRI for imaging of anatomical structures orblood flow does not require contrast agents as the varying properties ofthe tissues or blood provide natural contrasts. However, for morespecific types of imaging, exogenous contrast agents may beadministered. For a review of neural imaging techniques, see Mehrabianet al. (Front Oncol., 9:440 (2019).

In another aspect, a method is provided for detecting tau pathology. Themethod includes contacting a biological sample with an effective amountof a targeting ligand-liposome conjugate comprising a targeting ligandthat specifically binds to a cell surface marker of tau pathology,wherein the targeting ligand is conjugated to a liposome comprising adetectable label, washing the biological sample to remove unboundtargeting ligand liposome conjugate, and detecting tau pathology in thebiological sample by determining the amount of detectable labelremaining in the biological sample.

The targeting ligand-liposome conjugate can include any of the featuresdescribed herein. For example, in some aspects, the targeting ligand isan aptamer or stabilized aptamer, while in further aspects, thetargeting ligand is a thioaptamer. In yet further aspects, the aptameror stabilized aptamer used in the method comprises a DNA nucleotidesequence selected from, including selected from the group consisting ofTau_1 (SEQ ID NO: 5; DONGYBM), Tau_3 (SEQ ID NO: 6; MUSQD), Tau_9 (SEQID NO: 7), Tau_11 (SEQ ID NO: 8), Tau_10 (SEQ ID NO: 9), Tau_13 (SEQ IDNO: 10), Tau_8 (SEQ ID NO: 11), Tau_4 (SEQ ID NO: 12), Tau_17 (SEQ IDNO: 13), Tau_5 (SEQ ID NO: 14), Tau_21 (SEQ ID NO: 15), Tau_25 (SEQ IDNO: 16), Tau_7 (SEQ ID NO: 17), Tau_31 (SEQ ID NO: 18), Tau_42 (SEQ IDNO: 19), Tau_14 (SEQ ID NO: 20), Tau_19 (SEQ ID NO: 21), Tau_15 (SEQ IDNO: 22), Tau_56 (SEQ ID NO: 23), Tau_34 (SEQ ID NO: 24), Tau_23 (SEQ IDNO: 25), Tau_99 (SEQ ID NO: 26), and Tau_102 (SEQ ID NO: 27).

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers, and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. The level of detected labelcan be compared to control levels to determine if the biological sampleexhibits an increased level of cell surface markers for tau pathology.

Biological samples can be mammalian body fluids, sera such as blood(including whole blood, as well as its plasma and serum), CSF (spinalfluid), urine, sweat, saliva, tears, pulmonary secretions, breastaspirate, prostate fluid, seminal fluid, stool, cervical scraping,cysts, amniotic fluid, intraocular fluid, mucous, moisture in breath,animal tissue, cell lysates, tumor tissue, hair, skin, buccal scrapings,nails, bone marrow, cartilage, prions, bone powder, ear wax, etc., oreven from external or archived sources such as tumor samples (i.e.,fresh, frozen, or paraffin-embedded). Samples, such as body fluids orsera, obtained during the course of clinical trials may be suitable. Insome aspects, the biological sample comprises CSF or a sample containingneural cells, such as a neural (e.g., brain) tissue sample.

A biological sample may be fresh or stored. Samples can be stored forvarying amounts of time, such as being stored for an hour, a day, aweek, a month, or more than a month. The biological sample may beexpressly obtained for use or may be a sample obtained for anotherpurpose that can be subsampled. In some aspects, it may be useful tofilter, centrifuge, or otherwise pre-treat the biological sample toremove impurities or other undesirable matter that could interfere withanalysis of the biological sample.

In some aspects, the method includes the step of obtaining thebiological sample from a subject. The method of obtaining the biologicalsample will vary depending on the type of biological sample beingobtained, and such methods are well-known to those skilled in the art.For example, a sample of brain tissue can be obtained using asterotactic brain needle biopsy, while a sample of cerebrospinal fluidcan be obtained via a lumbar puncture.

ADx-002 Nanoparticles for MRI

For in vivo imaging of the hyperphosphorylative state in the brain oflive mice, two types of aptamer-targeted nanoparticles were developed asmolecular MRI contrast agents (ADx-002). One displayed the Tau_1 (SEQ IDNO: 5; DONGYBM) aptamer and the other displayed the Tau_3 (SEQ ID NO: 6;MUSQD) aptamer. PEGylated liposomal nanoparticles were synthesized usinga lipid mixture that included lipid-tethered Gd-DOTA for MR imaging andcholesterol for liposomal stability. Lipidized rhodamine was alsoincluded for studying ex-vivo microscopic distribution of liposomalnanoparticles in brain tissues using fluorescence microscopy. TheADx-002 compositions also included DSPE-mPEG2000 to increase the in vivocirculation time. Particles had a hydrodynamic diameter of ˜150 nm,˜86,000 Gd-chelates per liposome, and ˜500 aptamers conjugated to theouter leaflet of each liposomal nanoparticle (FIG. 4A).

In Vivo Molecular MRI Using ADx-002 for Detection ofHyperphosphorylative Cells

Studies were performed in P301S transgenic and age-matched wild typemice at 2-3 months of age. At this age, transgenic animals do not showfrank tau pathology (i.e., neurofibrillary tangles), but practically allwill develop tau pathology by around 8 months of age. Animals underwentbaseline, pre-contrast MRI. Thereafter, animals were intravenouslyadministered MM contrast agent (ADx-002—Tau_1 (SEQ ID NO: 5; DONGYBM) orADx-002—Tau_3 (SEQ ID NO: 6; MUSQD) or non-targeted control stealthliposomes. Delayed post-contrast MM was performed 4 days later. MRimages were acquired using a T1w-SE sequence and an FSE-IR sequence. Tgmice administered ADx-002—Tau_1 (SEQ ID NO: 5; DONGYBM) or ADx-002—Tau_3(SEQ ID NO: 6; MUSQD) demonstrated signal enhancement in the cortex andthe hippocampus regions of the brain (FIG. 5A). WT mice administeredADx-002—Tau_1 (SEQ ID NO: 5; DONGYBM) or ADx-002—Tau_3 (SEQ ID NO: 6;MUSQD) did not show signal enhancement in the brain. Similarly, Tg miceadministered non-targeted liposomal-Gd contrast agent did not showsignal enhancement in cortex or hippocampus. These regions of interestwere further analyzed quantitatively, and signal-enhancement between theTg and WT mice were found to be statistically significant (p<0.05) (FIG.5B).

A baseline enhancement threshold of ˜6% (=2× standard deviation ofsignal in baseline scans) was used as the classification threshold.Animals that showed signal enhancement above the threshold wereidentified as positives. ROC curves were generated with a six-pointordinal scale to assess sensitivity and specificity for detecting thegenotype, using ADx-002—Tau_1 (SEQ ID NO: 5; DONGYBM) or ADx-002—Tau_3(SEQ ID NO: 6; MUSQD) contrast agents, and constructed over the entiretested group, including controls. The aptamer-targeted nanoparticlecontrast agents, ADx-002— Tau_1 (SEQ ID NO: 5; DONGYBM) andrADx-002—Tau_3 (SEQ ID NO: 6; MUSQD), showed overall AUC and accuracy of˜0.95. ADx-002—Tau_3 (SEQ ID NO: 6; MUSQD) demonstrated highersensitivity than ADx-002—Tau_1 (SEQ ID NO: 5; DONGYBM) (FIG. 5C).

Post-mortem brain analysis was performed in 2-3 month old transgenic andwild-type mice. Immunofluorescence analysis using AT8 antibody stainingrevealed the presence of hyperphosphorylated tau species in transgenicmice but not in wild type mice (FIG. 5D). A 100% concordance wasobserved between AT8 positivity and animal genotype. In summary, in vivostudies demonstrated that ADx-002 enabled in vivo molecular Mill of thehyperphosphorylative state months before frank tau pathology, i.e., thepresence of neurofibrillary tangles, becomes evident in Tg mice.

Target Identification of Aptamers

To characterize the binding target of the aptamers, an aptamer-basedpulldown assay was performed, along with aptamer-basedimmunoprecipitation, followed by mass-spectrometry. The assay wasperformed for both Tau_1 (SEQ ID NO: 5; DONGYBM) and Tau_3 (SEQ ID NO:6; MUSQD) aptamers. A ranking of the abundance scores for identifiedproteins revealed keratin 6a, Keratin 6b, and VIM as possible bindingtargets. The surface expression of Keratin 6a, 6b was similar onwild-type and transgenic tissue sections, whereas the VIM expression washigher in the Tg mice (FIG. 6C). SH-SY5Y cells under undifferentiatedand differentiated hyperphosphorylative conditions show increasinglevels of VIM (FIGS. 6A, 6B), further suggesting it is a potentialtarget of aptamers Tau_1 (SEQ ID NO: 5; DONGYBM) and Tau_3 (SEQ ID NO:6; MUSQD) specific to the hyperphosphorylated state.

VIM Targeted WNP for MRI

Liposomal nanoparticles targeting VIM were prepared using Withaferin A,a small molecule that binds the conserved cysteine 382 residue ontetrameric VIM in a binding pocket that includes Gln 324 and Asp 331.DSPE-PEG3400-Withaferin A was synthesized and use do substitute for thecarboxy PEG in the aptamer targeted ADx-002 formulation to yield WNPwith ˜600 Withaferin A molecules per liposomal nanoparticle (FIG. 4B).Specific binding of WNPs to neuronally differentiated SH-SY5Y cellsunder hyperphosphorylative conditions is shown in FIG. 7.

Two month old P301S mice and APP/PSEN1 were injected with WNP and imagedusing the same T1-weighted sequences as used with the ADx-002nanoparticles. No signal enhancement was observed in the WT mice models,whereas the Tg mice (P301S and APP/PSEN1) showed distinct signalenhancement in the cortex and hippocampus regions and were identified aspositives (FIGS. 8A and 8B). Group statistical analysis (FIGS. 8B and8C) revealed that the VIM-targeted WNP contrast agents showed overallAUC and accuracy of ˜1.00. The phosphorylation status of tau intransgenic mice was confirmed by immunofluorescence (FIG. 9).

Alzheimer's Disease

In some aspects, the imaging indicates a level of tau pathologysufficient to diagnose the subject as having AD. In further aspects, themethod indicates that the subject has early stage AD, an increased riskof developing AD, or both. A level of tau pathology sufficient todiagnose the subject as having AD or early stage AD can be due to thepresence of increased levels of cell surface markers reflecting anincreased level of tau phosphorylation (e.g., hyperphosphorylation)within the cell (e.g., a neural cell). Examples of cell surface markersreflecting an increased level of tau phosphorylation include KRT6A,KRT6B, HSP, and VIM.

AD is a chronic neurodegenerative disease that usually starts slowly,gradually worsens over time, and is the cause of 60-70% of cases ofdementia. AD is characterized by loss of neurons and synapses in thecerebral cortex and certain subcortical regions. This loss results ingross atrophy of the affected regions, including degeneration in thetemporal lobe, parietal lobe, and parts of the frontal cortex andcingulate gyms. AD is a protein misfolding disease (proteopathy) causedby plaque accumulation of abnormally folded amyloid beta protein and tauprotein in the brain.

Diagnosis of AD is most often made in the moderate stage. Typically, thesymptoms of AD are cognitive dysfunction or deficiency and includedementia confirmed by medical and psychological exams, problems in atleast two areas of mental functioning, and progressive loss of memoryand other mental functions, especially where symptoms began between theages of 40 and 90, no other disorders account for the dementia, and noother conditions are present that may mimic dementia, includinghypothyroidism, overmedication, drug-drug interactions, vitamin B12deficiency, and depression. As the disease advances, symptoms caninclude problems with language, disorientation (including easily gettinglost), mood swings, loss of motivation, not managing self-care, andbehavioral issues. In some aspects, the methods and compositionsdescribed herein provide for the detection of early stage AD, which canbe present before one or more of these symptoms has manifested.Accordingly, in some aspects, the methods are used to diagnose a subjectthat does not exhibit any other symptoms of AD.

In some aspects, the methods further comprise providing prophylaxis ortreatment of AD to the subject. Prophylaxis of AD includes changes inlifestyle and diet that decrease the risk of developing AD.

Several medicines have also been identified that can be used to treatthe cognitive problems associated with AD. These includeacetylcholinesterase inhibitors such as tacrine, rivastigmine,galantamine, and donepezil, as well as the NMDA receptor antagonistmemantine. Huperzine A is a promising agent for treating AD, andatypical antipsychotics can be used for reducing aggression andpsychosis in people having AD.

Pharmaceutical Compositions

In some aspects, the compositions described herein are delivered as apharmaceutical composition. The pharmaceutical compositions are preparedaccording to standard techniques and may further comprise apharmaceutically acceptable carrier. Generally, normal saline will beemployed as the pharmaceutically acceptable carrier. Other suitablecarriers include, e.g., water, buffered water, isotonic solution (e.g.,dextrose), 0.4% saline, 0.3% glycine, and the like, includingglycoproteins for enhanced stability, such as albumin, lipoprotein,globulin, and the like. These compositions may be sterilized byconventional, well known sterilization techniques. The resulting aqueoussolutions may be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, and the like. Additionally, the liposome compositions can besuspended in suspensions that include lipid-protective agents thatprotect lipids against free-radical and lipid-peroxidative damages onstorage. Lipophilic free-radical quenchers, such as a-tocopherol andwater-soluble iron-specific chelators, such as ferrioxamine, aresuitable.

The concentration of liposome compositions in the pharmaceuticalformulations can vary widely, e.g., from less than about 0.05%, usuallyat or at least about 2-5%, to as much as 10 to 30% by weight, and willbe selected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. The amount of compositions administered will depend upon theparticular aptamer used, the disease state being treated, and thejudgment of the clinician. Generally, the amount of compositionadministered will be sufficient to deliver a therapeutically effectivedose of the nucleic acid. The quantity of composition necessary todeliver a therapeutically effective dose can be determined by oneskilled in the art. Typical dosages will generally be between about 0.01and about 50 mg nucleic acid per kilogram of body weight, between about0.1 and about 10 mg nucleic acid/kg body weight, or between about 2.0and about 5.0 mg nucleic acid/kg of body weight. For administration tomice, the dose is typically 50-100 μg per 20 g mouse.

Kits

In some aspects, kits are provided for preparing the liposomecomplexes/compositions. Such kits can be prepared from readily availablematerials and reagents, as described above. For example, such kits cancomprise any one or more of the following materials: liposomes, nucleicacid (condensed or uncondensed), hydrophilic polymers, hydrophilicpolymers derivatized with targeting ligands such as aptamers, andinstructions. A wide variety of kits and components can be prepared,depending upon the intended user of the kit and the needs of the user.For example, the kit may contain any one of the targeting moieties fortargeting the complex to a specific cell type, as described above.

Instructional materials for preparation and use of the liposomecomplexes can be included. While the instructional materials typicallycomprise written or printed materials, they are not limited to such. Anymedium capable of storing such instructions and communicating them to anend user is contemplated. Such media include, but are not limited to,electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. Such media mayinclude addresses to internet sites that provide such instructionalmaterials.

In various aspects, the instructions may direct a user to carry out anyof the method steps described herein. For example, the instructions maydirect a user to diagnose the risk that a subject will develop AD bydetecting the presence of tau pathology using the targeted liposomalcompositions described herein.

Examples have been included to describe more clearly particular aspectsof the invention. However, there are a wide variety of other aspectswithin the scope of the present invention, which should not be limitedto the examples provided herein.

EXAMPLES Materials and Methods

Cell-lines. SH-SY5Y cells (ATCC, Manassas, Va., #CRL-2266™) wereobtained from Dr. Jason Shohet's lab at the Texas Children's Hospital,Houston. Immortalized human hepatocytes (THLE-3) were purchased fromAmerican Type Culture Collection (ATCC, Manassas, Va., #CRL-11233™).Both were cultured according to the ATCC instructions. ReN Cell™ VM(#SCC008) was cultured as per instruction using neural stem cellmaintenance medium (#SCM005) and growth factors EGF (GF001) andbFGF(#GF005), all from Millipore Sigma, Burlington, Mass.

Differentiation.

SH-SY5Y cells were exposed to 30 μM all-trans-Retionic acid(Sigma-Aldrich, St. Louis, Mo., #R2625) in serum free cell medium for 10d with medium change every alternate day. ReNcell VM were differentiatedby the removal of growth factors from its culture medium for 10 d.

Hyperphosphorylation.

Hyperphosphorylation was induced in SH-SY5Y cells by addition of 30 nMOA (Sigma Aldrich, St. Louis, Mo., #459620) in growth medium with 30 μMRA for 24 h. ReNcell VM were hyperphosphorylated using 100 nM QA(SigmaAldrich, St. Louis, Mo., #P63204) in culture media for 24 h.

Synthesis of Primers and TA DNA Library.

All primers, Cy5, and amine labelled selected aptamers were purchasedfrom Integrated DNA Technologies (IDT, Coralville, Iowa). The ssDNAlibrary used in Cell-SELEX contained a central randomized sequence of 30nucleotides flanked by PCR primer regions to enable the PCRamplification of the sequence 5′-CGCTCGATAGATCGAGCTTCG (SEQ ID NO:28)—(N)₃₀—GTCGATCACGCTCTAGAGCACTG-3′ (SEQ ID NO: 29). The chemicallysynthesized DNA library was converted to a phosphorothioate modifiedlibrary by PCR amplification using, dATP (αS), resulting in the DNAsequences where the 3′ phosphate of each residue is substituted withmonothiophosphate groups. The reverse primer was labeled with biotin toseparate the sense strand from the antisense strand bystreptavidin-coated sepharose beads (PureBiotech, Middlesex, N.J.,#MSTR0510) for the next selection round. The concentration of the TAlibrary was determined with a NanoDrop™ 2000 by measuring the UVabsorbance at 260 nm.

Cell-SELEX.

The initial ssDNA library of 150 pmole was dissolved in binding bufferwith a total volume of 350 μl. It was denatured by heating at 95° C. for5 min and renatured by rapid cooling on ice for 10 min. The treatedSH-SY5Y cells at approximately 90% confluence in a 100-mm culture platewere washed twice with washing buffer and followed by incubating withthe ssDNA library of 150 pmole for 2 h at 4° C. Following theincubation, for positive selections, the supernatant was discarded, andcells were washed three times with washing buffer to remove any unboundsequences. Cells were scraped off and transferred to nuclease-freewater, following another 3× nuclease-free water washes. Cells innuclease-free water were centrifuged at 300×g for 5 min. QIAamp DNA Miniand Blood Mini kit (Qiagen, Germantown, Md., #51104) were introduced toelute cell membrane fraction. The cell membrane fraction wasPCR-amplified to monitor the presence of cell binding efficacy at eachcycle. For negative selections, the supernatant was simply pipetted outof the flask and processed for the next cycle of selection. The desiredcompartment was amplified by PCR and used to prepare the TA for the nextround of selection. Two different negative selections were involved. Onewas differentiated treatment only SH-SY5Y cells at cycles #12 and #13.Another was hepatocyte THLE-3 cells at cycles #20 and #21. A total of 26cycles of Cell-SELEX were conducted, including two different types ofnegative selections mentioned above.

Next-Generation Sequencing (NGS).

At the studied cycles, the membrane fractions were isolated, and therecovered TA sequences were amplified by PCR. Equimolar quantities ofthe recovered TA sequences over the range were pooled together andsequenced by Next-Gen DNA sequencing using Ion318 chip (ThermoFisher,Waltham, Mass.). A four base sequence was introduced during PCRamplification to serve as unique “barcode” to distinguish between thestudied cycles. Sequencing results were analyzed by the Aptalinger thatuses the markov model probability theory to find the optimal alignmentof the sequences.

Aptamer Binding Studies.

Aptamer binding studies were conducted with undifferentiated,differentiated, and hyperphosphorylated SH SY5Y and ReN cell VM grown in96-wells seeded at 10000 per well. Kd_(app) was measured by the equationY=Bmax X/(Kd+X), with GraphPad Prism 9, San Diego, Calif., with asaturation binding experiment; cells were incubated with varyingconcentrations of Cy5-labeled aptamer in a 100 μl volume of bindingbuffer containing cells, incubated for 30 min, washed twice, resuspendedin 100 μl buffer, and analyzed by a Molecular probes microplate readerequipped with the appropriate excitation and emission filters. All datapoints were collected in triplicate.

Immunocytochemistry.

An eight-well glass plate was coated with a solution of 100 μg/mlCollagen Type I (Thermofisher Scientific, Waltham, Mass., #A1064401)dissolved in 0.01N HCl and air dried, PBS washed, and air dried againprior to seeding with 20,000 SH-SY5Y cells per well. Aptamer staining at100 nM was performed with live cells for 2 h at 4° C. in binding bufferand washed twice with washing buffer. Cells were fixed by incubation for15 min in 4% formaldehyde in PBS at rt. Non-specific binding was blockedwith blocking buffer (G-Biosciences, St. Louis, Mo., #786195) for 1 h,and overnight incubation at 4° C. with the rabbit pTau primary antibody(1:100) (Santa Cruz Biotechnology, #: sc-101815) was followed by washingwith PBS, and 1 h incubation with goat anti-rabbit IgG secondaryantibody, Alexa Fluor 488 (Invitrogen, Carlsbad, Calif., #A-11008) for 1h at rt. Cytoskeletal actin filaments were stained with Alexa Fluor 594Phalloidin (Invitrogen, #A12381). The cells were covered withVECTASHIELD hardset mounting medium with DAPI (Vector Laboratories,Burlingame, Calif., #H-1500) for 5 min at rt. Images were visualizedunder Olympus Fluoview FV1000 confocal microscopy.

ADx-002 Nanoparticle Synthesis.

L-α-phosphatidylcholine, hydrogenated (Hydro Soy PC; HSPC) andCholesterol were purchased from Lipoid Inc., Newark N.J., USA.1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-mPEG2000) was purchased from Corden Pharma,Liestahl, Switzerland. DSPE-PEG3400-COOH and Gd-DOTA-DSPE weresynthesized in house, lis-rhodamine-DHPE from ThermoFisher Scientific.HSPC, Cholesterol, DSPE-PEG3400-COOH, DSPE-mPEG2000, Gd-DOTA-DSPE,lis-rhodamine-DHPE at molar proportions 31.4:40:0.5:3:25:0.1 weredissolved in ethanol to achieve a total concentration of 100 mM. For thenon-targeted control stealth liposomes, carboxy terminated PEG was notincluded in the lipid mixture. The ethanolic solution of lipids washydrated with 150 mM saline solution at 65° C. for 30 min, allowingmultilamellar liposomes to form. The mixture was then extruded in a 10ml Lipex extruder (Northern Lipids Inc., Burnaby, Canada) using a 400 nmpolycarbonate track-etch polycarbonate filter (3 passes) followed by a200 nm (3 passes) and finally 100 nM filters. The suspension wasdiafiltered using a MicroKros cross-flow diafiltration cartridge (500kDa cutoff) from Repligen, Rancho Dominguez, Calif., exchanging theexternal buffer for phosphate buffered saline (PBS, pH 7.2) for 15volume exchanges. To form the aptamer conjugated liposomes, liposomeswith lipid-PEG-COOH were reacted with amine terminated aptamers usingcarbodiimide chemistry. The carboxyl groups on the liposomes wereactivated with 5 mM EDC and 10 mM sulfo-NHS at pH˜6 for 5-10 min. Theactivated liposomes were then immediately reacted with the amineterminated aptamers, and the pH was raised to ˜7.3-7.6 by titrating μlamounts of 5 N NaOH. The final concentration of aptamers used inreaction is ˜140 μM. The reaction was mixed at rt for 1 h followingwhich the reaction was carried out at 4° C. overnight. The liposomeswere dialyzed against PBS to remove unconjugated aptamers using a 300kDa dialysis membrane. The dialysate (external phase) was concentratedusing 10 kD centrifugal separator and washed with PBS to remove residualEDC/s-NHS. The concentrated dialysate was analyzed by NanoDropSpectrophotometer (ThermoFisher Sci., Waltham, Mass., USA) to determineunconjugated aptamer fraction and estimate aptamer density pernanoparticle in ADx-002 formulations. Inductively coupled plasma atomicemission spectroscopy (ICP-AES) was used to measure Gd and phosphorusconcentrations of ADx-002 formulations. The hydrodynamic diameter ofliposomal nanoparticles in ADx-002 formulations was determined using adynamic light scattering instrument.

Withaferin a Nanoparticle (WNP) Synthesis.

The allylic alcohol of Withaferin A was selectively activated byexposure to 4-nitrophenyl chloroformate (1.1 eq) at 0° C. for 8 h togive intermediate compound in excellent yield. This was then reactedwith DSPE-PEG-NH2-3400 at rt for 24 h. The crude product was dialyzedagainst water for 2 days and freeze dried to yieldDSPE-PEG-3400-Withaferin A. Structures of the intermediate and finalproducts were confirmed by NMR and MALDI. Liposomal nanoparticlescontaining Withaferin A on its surface were generated by using theADx-002 composition, substituting the carboxy PEG with the synthesizedDSPE-PEG3400-Withaferin A to yield WNP (FIG. 4B).

Mice.

All of the procedures were performed with approval from InstitutionalAnimal Care and Use Committee (IACUC) of Baylor College of Medicine.Mice were kept under a 12 h light/dark cycle, with food and wateravailable ad libitum. PS19 mice from Jackson Laboratories (Bar Harbor,Me.) B6; C3-Tg (Prnp-MAPT*P301S) PS19Vle/J Stock No: 008169 were used,and experiments were conducted at 2 months of age. The Tg mice developneurofibrillary tangles by 5 months of age. Age-matched non-transgenicWT mice were used as controls. APP/PS1 mice from Jackson LaboratoriesB6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax MMRRC Stock No: 34832-JAX were alsoused for experiements with WNP. These mice generate amyloid plaques by 6weeks in cortex and 2-4 months in hippocampus without any reportedmature tau tangles, but the presence of hyperphosphorylated tau neuriticprocesses has been observed around plaques.

Magnetic Resonance Imaging (MRI).

MRI was performed on a 1T permanent magnet scanner (M7, Aspect Imaging,Shoham, Israel). Mice underwent pre-contrast baseline scans. Thereafter,mice were intravenously administered one of three nanoparticle MRcontrast agents (Tau_1 (SEQ ID NO: 5; DONGYBM), Tau_3 (SEQ ID NO: 6;MUSQD), or non-targeted control liposomes) via tail vein at a dose of0.15 mmol Gd/kg of body weight. Delayed post-contrast MRI was performed4 days after contrast agent injections. Pre-contrast and delayedpost-contrast MR images were acquired using a T1w-SE sequence and anFSE-IR sequence with the following parameters: SE parameters: TR=600 ms,TE=11.5 ms, slice thickness=1.2 mm, matrix=192×192, FOV=30 mm,slices=16, NEX=4; FSE-IR parameters: TR=13500 ms, TE=80 ms, TI=2000 ms,slice thickness=2.4 mm, matrix=192×192, FOV=30 mm, slices=6, NEX=6. Coilcalibration, RF calibration, and shimming were performed at thebeginning of study for each subject. The pre-contrast scans provide abaseline for calculation of signal enhancement from resultingpost-contrast scans. Two-standard deviations above the mean variationwithin WT control animals was used as the cutoff signal intensity foridentifying tau positive animals. Six Tg mice and six WT mice were usedfor testing of each nanoparticle contrast agent formulation. ROC curveswere generated on a six point ordinal scale by plotting the TPF againstthe FPF based on imaging-based identification of tau-positive animalsusing the cutoff signal intensity and comparing against histologicalconfirmation of tau pathology as a gold standard. A fitted curve wasgenerated against the empirical points plotted on the graphs.Qualitative and quantitative analysis of MRI images was performed inOsiriX (version 5.8.5, 64-bit, Pixmeo SARL, Geneva, Switzerland) andMATLAB (version 2015a, MathWorks, Natick, Mass.).

Immunohistochemistry.

After the final Mill scan, the mice were euthanized and perfusedextensively with 0.9% saline followed by 4% paraformaldehyde for 15 min.The brains were then immersion-fixed in 4% formaldehyde for 48 h at 4°C., transferred to 30% sucrose for cryoprotection, and embedded in OCT.Phenotypic confirmation for the presence of phosphorylated tau and VIMwas performed on 25 μm thick brain sections. Antigen retrieval in pH=8.5citrate buffer was executed in a 1200W GE microwave for 15 min. After 15min of cooling, 25 μL of 1:50 dilution of primary p-tau antibody, namelyeither ATB, AT100, or AT180, which recognize different p-tau species,were incubated in a tray (RPI, Mt. Prospect, Ill. #248270) designed formicrowave enhanced immunostaining procedures for 3 min at power level 3.After a 2 min cooling, sections were washed with PBS and incubated for 3min with a 1:100 dilution of appropriate secondary antibody. DAPIstaining proceeded after 2 min of cooling and a PBS washing. ProGoldAntifade (Invitrogen, Carlsbad, Calif., #P36030) was used to mountslides, which were visualized on Olympus Fluoview LV100. Scanning ofwhole sections was also conducted using a Biotek Cytation 5 slidescanning microscope. Antibodies—AT8 (#MN1020) and Vimentin SP20(#MA516409) were purchased from Thermo Fisher Scientific, Waltham,Mass., Vimentin D21H3 from Cell Signaling Technology, Beverly, Mass.,#5741T, and cell-surface vimentin from Abnova, Taipei City, Taiwan,#H00007431-M08J.

Target Identification.

The protein targets of Tau_1 (SEQ ID NO: 5; DONGYBM), Tau_3 (SEQ ID NO:6; MUSQD), Tau-4, and Tau-5 were identified by affinity-pull down usingthe selected aptamers as the capturing reagent followed bymass-spectroscopy. A scrambled DNA sequence, R2, was used as a control.The hyperphosphorylated SH-SY5Y cells, at 90-95% confluence, were washedwith cold PBS buffer and incubated with biotinylated selected aptamerswith 25 mmol/l each at 4° C. in PBS, respectively. After 2 hours ofgentle agitation, SH-SY5Y cells were cross-linked with 1% formaldehydefor 10 min at rt. The formaldehyde cross-linking was quenched withglycine. Cells were scraped from the plate, washed, lysed with lysingbuffer (Thermofisher Scientific, #87787), and treated with proteaseinhibitor mixture. The lysates were freeze-thawed for 30 min on ice andcleared by centrifuging at 10,000×g for 2 min at 4° C. To pull down thecross-linked proteins, equal amounts of cell lysate were incubated withprewashed streptavidin magnetic beads for 1 h at rt with continuousrotation. Protein digestions were performed on the beads to isolatetargeted proteins and processed for mass spectrometric analysis. Eachsample was analyzed in triplicate. The raw data files were processed togenerate a Mascot Generic Format with Mascot Distiller and searchedagainst the SwissProt_2012_01 (Human) database using the licensed Mascotsearch engine v2.3.02 (Matrix Science, Boston, Mass.) run on an in-houseserver.

The ATN research framework suggests the need for biomarkers to diagnoseand classify AD. Under this framework, CSF based detection of AP and tau(total, and phosphorylated) have been reported but only at the prodromalstage of disease, in patients with mild cognitive impairment.Non-invasive neuroimaging tools, such as structural MRI, to diagnose andmonitor neurodegeneration, show a definitive correlation with cognitivedecline, visualizing atrophic regions that depict neuronal injury inlate stage disease. However, a reliable marker of early stage disease inthe presymptomatic stage is yet to be described.

While the role of AP and tau in the development of AD, and the mechanismof transition from presymptomatic to symptomatic AD are yet unclear, thetime scale of the transition is generally accepted to be over a periodof 10-20 years. AP deposits are considered the start ofneurodegeneration, but recent studies indicate that tau pathology showsa stronger correlation with disease progression suggesting that thelimitation of current tests is their inability to identify early stagepathological tau. CSF presence of hyperphosphorylated tau species p-181and p-217 is associated with AP deposition that precedes a positive tauPET but only has a concordance of 50%-70%. Taken as a whole, the rolesof AP and tau deposition in disease progression, and the role of AP inthe spread of initial tau aggregates, strongly suggest that a biomarkerof pathological tau at a presymptomatic stage of the disease is likelyto advance detection by several years and constitutes the motivation forthis work.

Initial tau aggregation is thought to be triggered by an imbalance incellular homeostasis caused by dysregulated phosphorylation. Severalkinases can phosphorylate tau, theoretically at 85 different positionsof which at least 45 have been observed experimentally. Combined withreduced phosphatase activities in AD, the altered kinase-phosphatasebalance yields hyperphosphorylative conditions that cause abnormalhyperphosphorylation of tau. Disruption of the normal function of tau,modulating microtubule dynamics by lowering its binding capabilities,increases the level of cytosolic free tau leading to aggregation andfibrillization of tau that spreads throughout the connected brain,seeding the pathology. This initial process of hyperphosphorylation maybe associated with changes on the surface of hyperphosphorylative cells.Through the instant work, these surrogate markers of tauhyperphosphorylation that presage future tau pathology have beenidentified.

Using SH-SY5Y cells as a model of neuronal hyperphosphorylation, an RPPAanalysis was used to demonstrate elevated levels of surface moleculesspecific to the hyperphosphorylative state. Cell-SELEX capturing thedifferences between the surface of hyperphosphorylative cells and normalcells allowed the selection of phosphorothioate modified short DNAaptamers that bound with high affinity and specificity tohyperphosphorylative cells (FIG. 2A). Having identified unique aptamersthat bound such markers, MR molecular imaging contrast agents weredeveloped that recognize the surface of cells in hyperphosphorylativestate. SH-SY5Y cells are not true neurons, but rather, a cell lineoriginating in a neuroblastoma, a tumor of embryological neural crestorigin. However, they can be induced to differentiate to a neuronalphenotype (as in the current work). While primary neuronal culture orimmortalized neuronal cells, e.g., ReN-VM, may offer alternative modelsof neurons, the aptamer hits from the SELEX screen have beenfunctionally tested in a Tg mouse model of tau deposition. Theirperformance has been validated, supporting the position that the choiceof cell model was adequate to identify suitable markers of tauhyperphosphorylation.

The possible binding targets of the aptamers have been narrowed down,and the data suggest that cell surface VM is a likely target. Thespecific presence of cell surface VIM on the surface of SH-SY5Y cells ina hyperphosphorylative state and on P301S mouse brain sections has beenconfirmed. VIM is an intermediate filament protein that undergoesconstant assembly and/or remodeling and is usually associated withmesenchymal cells. The assembly state of filaments is linked to theirphosphorylation state; phosphorylation promotes disassembly. VIMcontains more than 35 phosphorylation sites targeted by multiple kinasesand phosphatases, allowing it to adjust IF dynamics dependent on itsenvironment. Mechanical, chemical (toxins, hypoxia), and microbialstresses upregulate VIM, and its phosphorylation allow cells to adjusttheir mechanical properties. The balance of different oligomeric formsinfluence dynamic cell processes including adhesion, migration, andinvasion, including stress-induced signaling. Vim IFs (˜10 nm)distributed throughout the cell by association with microtubules(tubulin, 24 nm) regulating cell-migration, and microfilaments (actin, 7nm) regulating cell-contractility, form the cytoskeletal network andprovide mechanical support for the plasma membrane where it contactsother cells or the extracellular matrix. During the biological process,epithelial to mesenchymal transition, wherein non-motile, polarepithelial cells transform to motile invasive non-polar mesenchymalcells, cells also undergo a cytoskeletal reorganization that includeschanges in cell-membrane integrity, disassembly of junction proteins,increased stress-fiber formations, and altered cell-surface proteinexpression. Changes in the localization of proteins is a hallmark ofthis pathologic process. The observation that VIM is upregulated andtranslocated to the cell surface in the early stages of tauhyperphosphorylation suggests a possible role for EMT-related processesat the start of a slow progression towards AD pathology.

PET is the leading modality for clinical molecular imaging, driven byits high contrast sensitivity; however, it suffers from poor spatialresolution on the order of 5-10 mm, high cost, limited access toradioactive tracers, and radiation exposure. Nanoparticle-enhanced MRimaging overcomes all these obstacles, but historically has not achievedhigh enough sensitivity. Liposomal nanoparticles have been demonstratedexhibiting large numbers of Gd chelates in the external bilayer leaflet,with hyper-T1 relaxive properties resulting in contrast sensitivity thatrivals nuclear imaging.

An often-quoted concern about the use of nanoparticles for brain imagingcenters on whether these particles can penetrate the blood-brain barrier(BBB). The notion of the BBB arose primarily in the context of deliveryof relatively large amounts of therapeutic molecules to brain tumors.For imaging, however, relatively small amounts of contrast agent need tobe delivered. Further, the convective and diffusive transport ofmolecular and particulate species through the porous choroid plexus iswell known. The transport of liposomal nanoparticles into the CSF viathis route has been demonstrated. A liposomal MM agent targeting amyloidplaque that successfully traverses the BBB following intravenousinjection and binds plaques, enabling accurate imaging of amyloidpathology by T1-weighted MM has been demonstrated.

In P301S mice, the earliest reported histopathological studies are atthe age of 2.5 months and report no tau pathology. “Tau seeding” thecell-cell transfer of pathogenic tau aggregates has been reported usingbrain homogenates at 1.5 month of age. Therefore, P301S mice at 2 monthof age were chosen for these studies, an age at which tau seeding shouldbe taking place, but frank tau pathology should be absent. The mice wereinjected with ADx-002 nanoparticles targeted either by the Tau_1 (SEQ IDNO: 5; DONGYBM) aptamer or the Tau_3 (SEQ ID NO: 6; MUSQD) aptamer. Whenimaged by T1-weighted Mill sequences, designed to optimize signal fromthe Gd chelate induced T1 relaxation caused by the liposomal-Gdnanoparticles, signal enhancement was observed in the cortex andhippocampus regions of the brain. Hyperphosphorylative conditions wereconfirmed by post-mortem IF staining with AT8 antibody that recognizesthe 5202 and T305 pTau species. Signal enhancement was not observed innon-Tg mice or in Tg mice injected with untargeted nanoparticles,supporting the specificity of Tau_1 (SEQ ID NO: 5; DONGYBM) and Tau_3(SEQ ID NO: 6; MUSQD)-bearing nanoparticle binding to target.

Validation is herein provided of VIM binding ADx-002 nanoparticles bythe use of a small molecule, Withaferin A, known to bind VIM at itshighly conserved cysteine residue in coiled-coil 2B domain.Intraveneously injected WPNs exhibited binding in the same brain regionsas ADx-002 and maintained specificity and sensitivity to thephosphorylative state, with signal enhancement observed only in Tg miceand not in WT mice. Additionally, similar results are shown in anothermice model (APP/PSEN1). Taken together, this in vivo data furthersupports the use of such particles as detectors of hyperphosphorylationthat leads to the initiation of tau pathology in AD. The higherexpression of CSV was confirmed in transgenic P301S mice at 2 months ofage by immunofluorescence (FIG. 10).

While PET is the mainstay of molecular imaging and exhibits remarkablesensitivity, there are several limitations posed by this methodology.Access to PET imaging is limited, even in the relatively well-servedU.S., and is skewed toward high density urban centers. PET costs arevery high due to the need for same day radiosynthesis and rapid decay ofthe isotopes. Longer half-life isotopes cause higher radiation exposure.This tradeoff between half-life and radiation exposure greatly limitsthe reach of PET to a wider patient population. Current PET tau tracersrecognize the tau (3-sheets in the PHF and NFT present in tauopathies.This conformation is not unique to tau, and the in vivo specificity iscircumspect, limiting its interpretation. Off-target binding ofFlortaucipir, an approved tau PET agent, has been reported since itbinds the MAO-B enzyme in the brain. Further, the vast majority ofpathological tau is intracellular, posing a significant barrier to PETtracers that must navigate to the site of tau pathology, bind thetarget, and have all unbound tracer molecules cleared from the brainbefore the radioactive signal decays. The choice of MRI as the detectionmodality is based on hyper-T1 relaxive properties of nanoparticles withsurface conjugated Gd chelates, bringing detection sensitivity to thesame range as nuclear imaging, and the MM agent does not suffer from therapid signal decay of PET agents, allowing plenty of time for unboundtracer to clear from the brain before imaging. The choice of a cellsurface surrogate marker of tau hyperphosphorylation avoids the need tobind an intracellular target. Finally, MRI imaging is already includedin AD management and can be adjusted with agents such as ADx-002nanoparticles to constitute a highly sensitive and specific test forfuture tau pathology.

What is claimed is:
 1. A composition for identifying tau pathology, comprising a targeting ligand that specifically binds to a cell surface marker of tau pathology, wherein the targeting ligand is linked to a liposome comprising an imaging agent.
 2. The composition of claim 1, wherein the targeting ligand comprises an aptamer.
 3. The composition of claim 1, wherein the cell surface marker of tau pathology comprises a cell surface marker of tau hyperphosphorylation.
 4. The composition of claim 1, wherein the targeting ligand is determined to specifically bind to a cell surface marker of tau pathology using a systematic evolution of ligands by exponential enrichment (SELEX) method.
 5. The composition of claim 1, wherein the targeting ligand comprises a DNA nucleotide sequence selected from the group consisting of Tau_1 (SEQ ID NO: 5), Tau_3 (SEQ ID NO: 6), Tau_9 (SEQ ID NO: 7), Tau_11 (SEQ ID NO: 8), Tau_10 (SEQ ID NO: 9), Tau_13 (SEQ ID NO: 10), Tau_8 (SEQ ID NO: 11), Tau_4 (SEQ ID NO: 12), Tau_17 (SEQ ID NO: 13), Tau_5 (SEQ ID NO: 14), Tau_21 (SEQ ID NO: 15), Tau_25 (SEQ ID NO: 16), Tau_7 (SEQ ID NO: 17), Tau_31 (SEQ ID NO: 18), Tau_42 (SEQ ID NO: 19), Tau_14 (SEQ ID NO: 20), Tau_19 (SEQ ID NO: 21), Tau_15 (SEQ ID NO: 22), Tau_56 (SEQ ID NO: 23), Tau_34 (SEQ ID NO: 24), Tau_23 (SEQ ID NO: 25), Tau_99 (SEQ ID NO: 26), and Tau_102 (SEQ ID NO: 27).
 6. The composition of claim 5, wherein the targeting ligand comprises the DNA nucleotide sequence Tau_1 (SEQ ID NO: 5).
 7. The composition of claim 5, wherein the targeting ligand comprises the DNA nucleotide sequence Tau_3 (SEQ ID NO: 6).
 8. The composition of claim 1, wherein the cell surface marker of tau pathology comprises a protein selected from KRT6A, KRT6B, HSP, and VIM.
 9. The composition of claim 1, wherein the imaging agent comprises a magnetic resonance imaging (MM) contrast enhancing agent.
 10. The composition of claim 1, wherein the liposome comprises a membrane, the membrane comprising: a first phospholipid; a sterically bulky excipient that is capable of stabilizing the liposome; a second phospholipid that is derivatized with a first polymer; a third phospholipid that is derivatized with a second polymer, the second polymer being conjugated to the targeting ligand; and the imaging agent, which is encapsulated by or bound to the membrane.
 11. The composition of claim 10, wherein: the first phospholipid comprises HSPC; the sterically bulky excipient that is capable of stabilizing the liposome comprises cholesterol; the second phospholipid that is derivatized with a first polymer comprises DSPE-PEG; the third phospholipid that is derivatized with a second polymer, the second polymer being conjugated to the targeting ligand comprises DSPE-PEG conjugated to at least one of Tau_1 (SEQ ID NO: 5) and Tau_3 (SEQ ID NO: 6); and the imaging agent, which is encapsulated by or bound to the membrane comprises DSPE-DOTA-Gd.
 12. The composition of claim 10, wherein: the first phospholipid comprises HSPC; the sterically bulky excipient that is capable of stabilizing the liposome comprises cholesterol; the second phospholipid that is derivatized with a first polymer comprises DSPE-PEG2000; the third phospholipid that is derivatized with a second polymer, the second polymer being conjugated to the targeting ligand comprises DSPE-PEG3400 conjugated to at least one of Tau_1 (SEQ ID NO: 5) and Tau_3 (SEQ ID NO: 6); and the imaging agent, which is encapsulated by or bound to the membrane comprises DSPE-DOTA-Gd.
 13. The composition of claim 12, wherein the ratio of HSPC:Cholesterol:DSPE-mPEG2000:DSPE-PEG3400:DSPE-DOTA-Gd=about 31.5:about 40:about 3:about 0.5:about
 25. 14. The composition of claim 12, further comprising about 250-500 molecules of conjugated Tau_1 (SEQ ID NO: 5).
 15. The composition of claim 12, further comprising about 150-400 molecules of conjugated Tau_3 (SEQ ID NO: 6).
 16. A targeting composition, comprising a phospholipid linked to a polymer that is linked to a targeting ligand that specifically binds to a cell surface marker of tau pathology.
 17. The targeting composition of claim 16, wherein the targeting ligand comprises an aptamer.
 18. The targeting composition of claim 16, wherein the targeting ligand comprises a DNA nucleotide sequence selected from the group consisting of Tau_1 (SEQ ID NO: 5), Tau_3 (SEQ ID NO: 6), Tau_9 (SEQ ID NO: 7), Tau_11 (SEQ ID NO: 8), Tau_10 (SEQ ID NO: 9), Tau_13 (SEQ ID NO: 10), Tau_8 (SEQ ID NO: 11), Tau_4 (SEQ ID NO: 12), Tau_17 (SEQ ID NO: 13), Tau_5 (SEQ ID NO: 14), Tau_21 (SEQ ID NO: 15), Tau_25 (SEQ ID NO: 16), Tau_7 (SEQ ID NO: 17), Tau_31 (SEQ ID NO: 18), Tau_42 (SEQ ID NO: 19), Tau_14 (SEQ ID NO: 20), Tau_19 (SEQ ID NO: 21), Tau_15 (SEQ ID NO: 22), Tau_56 (SEQ ID NO: 23), Tau_34 (SEQ ID NO: 24), Tau_23 (SEQ ID NO: 25), Tau_99 (SEQ ID NO: 26), and Tau_102 (SEQ ID NO: 27).
 19. An aptamer or stabilized aptamer comprising a DNA nucleotide sequence selected from the group consisting of Tau_1 (SEQ ID NO: 5), Tau_3 (SEQ ID NO: 6), Tau_9 (SEQ ID NO: 7), Tau_11 (SEQ ID NO: 8), Tau_10 (SEQ ID NO: 9), Tau_13 (SEQ ID NO: 10), Tau_8 (SEQ ID NO: 11), Tau_4 (SEQ ID NO: 12), Tau_17 (SEQ ID NO: 13), Tau_5 (SEQ ID NO: 14), Tau_21 (SEQ ID NO: 15), Tau_25 (SEQ ID NO: 16), Tau_7 (SEQ ID NO: 17), Tau_31 (SEQ ID NO: 18), Tau_42 (SEQ ID NO: 19), Tau_14 (SEQ ID NO: 20), Tau_19 (SEQ ID NO: 21), Tau_15 (SEQ ID NO: 22), Tau_56 (SEQ ID NO: 23), Tau_34 (SEQ ID NO: 24), Tau_23 (SEQ ID NO: 25), Tau_99 (SEQ ID NO: 26), and Tau_102 (SEQ ID NO: 27).
 20. The aptamer or stabilized aptamer of claim 19, wherein the DNA nucleotide sequence is positioned between SEQ ID NO: 1 and SEQ ID NO:
 2. 